BIoenergetics BTOL Cramer 25 9 07

BTOL-Bioenergetics 1
Chapter One
1. Thermodynamics of Energy Transduction in Biological
Membranes
W. A. Cramer and G. M. Soriano
1-1. Introduction
Thermodynamics is a classical area of physics and chemistry for which there exists a large
number of major treatises that form the background for this chapter. These include excellent
treatises that emphasize the application of thermodynamics to problems in biochemistry and
biophysics (Bloomfield, Biophysics Textbook On-Line ; Eisenberg and Crothers, 1979; Tinoco et
al., 1978). Other sources are (Hill, 1966; Mahan, 1964; Chandrasekhar, 1957). The present
treatment specifically emphasizes the application of thermodynamics to bioenergetics, or energy
transduction in biological membranes. This energy transduction involves (i) the transduction of
light and redox energy to "free energy" stored in a trans-membrane ionic electrochemical potential
(∆µ~), and (ii) the use of this energy to synthesize ATP and to drive active transport across
membranes and the accumulation of needed solutes and metabolites in the cell. This present
discussion of a subset of the topics of thermodynamics in the context of biological membrane
energy transduction is derived from that in (Cramer and Knaff, 1991).
The laws of thermodynamics, obeyed by physical and chemical reactions in living as well as
inanimate systems, describe the energetic limitations on state changes of systems of molecules, the
relative molecular populations of energetically accessible states, and predict the direction of the
reactions that link these states. In the thermodynamic formalism, a "system" is described physically
and mathematically by a small number of state properties or variables: (a) intensive variables
such as the density, dielectric constant, molar free energy or chemical potential, pressure, specific
heat, and temperature, which are independent of the size of the system; (b) extensive variables,
such as the mass, energy, enthalpy, and entropy, and volume, which are dependent on system size.
The state of a system is said to be defined when all of its properties can be specified. A defined
state of a system is in equilibrium when (a) the values of its state variables are independent of
time and (b) there is no flux of mass or energy across the boundaries of the system. If the system
variables are constant, but there is a net flux of mass or energy moving across the system, then this
system is not in equilibrium, but in a steady-state. Mathematical relationships between state
variables are called equations of state. One example of such an equation, relating the pressure, p,
volume, V, and temperature, T, is the ideal (dilute) gas equation,
pV = nRT (1-1)
where the constants of proportionality, n, R, are, respectively, the number of moles in the system and
the gas constant, R (Table 1-1, Constants and Conversions). This equation of state applies to gases
at a pressure and concentration small enough that molecular interactions can be neglected. The
same equation of state, with the variable, concentration (c), replacing that of pressure, can be used as
an equation of state for solutes in dilute solution, so that the dependence of the energy change of an
ideal gas at constant temperature on its change of pressure, dE = Vdp, is the same as the
dependence of the partial free energy of a solute in aqueous solution on its concentration, dG = Vdc
(cf., section 1-7 below.)
A summary of physical constants that are utilized in discussions of thermodynamics, their
values in the Standard International (SI) and cgs (cm-gm-sec) systems of units, and the conversion
factors between the two systems, is presented in Table 1-1.
Copyright by Cramer and Soriano, 2002

Table 1-1. Constants and Conversions

A. Fundamental Constants
Constant Symbol SI Units Value cgs Units
Avogadro’s number N 6.02 x 1023 mol–1

Speed of light in vacuum c 3·108 m s–1 3·1010 cm s–1
Bottzmann’s constant k 1.38·10–23 J K–1 1.38·10–16 erg K–1
Gas constant (Nk) R 8.31 J K–1 mol–1 1.99 cal K–1 mol–1
Elementary charge e 1.6·10–19 coulomb 4.8 x 10–10 statcoulomb
(g1/2cm3/2 s–1)
Faraday constant (Ne) F 9.65·104 C mol–1
Planck’s constant h 6.63·10–34 J s 6.63·10–27 erg s
h= h
2π
B. Inter-Conversion of Units
1 Joule (SI) = 107 ergs (cgs) = 1 coulomb-volt
1 electron-volt (eV) = 1.6 · 10–19 J = 23.06 kcal · mol–1 = 96.48 kJ mol–1
1 calorie = 4.184 Joule
1 ampere = 1 coulomb · s–1 = 6.2·1018 charges · s–1
1 Siemen = 1 amp/volt
1 Einstein = 1 mole of photons
C. Useful Mixed Constants

2.3 RT = 1.36 kcal · mol–1 = 5.69 kJ mol–1 (25oC)
2.3 RT/F = 59.1 mV (25oC)
Thought Problems:
1. Consider two systems: (a) hemoglobin and O2, each at a constant total concentration, in a sealed
test tube at constant temperature (isothermal) and pressure (isobaric), where the binding of O 2 is
described by Hb + O 2 ← → Hb-O2; (b) a suspension of bacterial cells in a solution of nutrient
(“chemostat”), amino acids, and salts in which the cells are diluted with fresh nutrient medium so
they remain at a constant cell concentration. Which system, (a) or (b), is at equilibrium and which
is at steady-state?
2. Consider the heat energy absorbed by a protein when it is heated at constant pressure in a
scanning calorimeter from room temperature, 20° C, to its melting temperature, Tm = 60° C. The
-1 -1
average specific heat, Cp, of the protein in this temperature range is 40 kJ mol ˚K . For what
extensive variable is a value needed to calculate the total amount of absorbed heat energy ?
3. The hydrophobic interior of biological membranes is an energy barrier for transport across the
membrane of charged ions and metabolites. The hydrophobicity of the membrane bilayer semi-
continuum is described by the dielectric constant, ε, which describes the polarity of the medium, and
has the value of 78 in water, and of approximately 3 in the center of a lipid bilayer membrane. The
energy required to transfer an ion of radius, r, and valence, z, from water with dielectric constant, εw,
to the center of the membrane with dielectric constant, εm, is the energy, Eb = 695(z)2/r [1/εm - 1/εw],

in kJ/mol, that forms a barrier for the translocation of the ion to the membrane interior. What is the
physical constant (Table 1-1) that allows one to calculate the energy barrier per ion?
1-2. The First Law of Thermodynamics

A fundamental aspect of the First Law, which states that the energy of an insulated
system is conserved, is that heat and work are both considered as equivalent forms of
energy. The energy level of a system can be changed by (i) an increment or decrement of
mechanical work (dW), exerted by a force (F) or pressure (P) acting, respectively, over a distance
dx, or an element of volume, dV, or by (ii) heat (dQ) transmitted through a temperature gradient.
The First Law, the law of conservation of energy, is well known from other branches of
chemistry and physics and states that the total energy of a system insulated from its surroundings
does not change. Thus, addition of amounts of heat (dQ) and work (dW) to a system that is
insulated from exchange of energy with its surroundings must be reflected in a change, dE, of the
energy of the system. That is,
dE = dQ + dW (1-2)
1-2.1 Properties of the energy, E:

1. E is an extensive property, whose units in thermodynamic problems are calories or joules
(1 cal = 4.184 J or electron-volts (1 eV = 1.6 x 10-19 J).
• For an ideal or dilute gas, the energy is a function only of the intensive variable, temperature.
• Energy is a state function. The value of the energy function is unique for a given state, and
therefore its change, ∆E, between two states is independent of the pathway that links the states.
Because of the latter two properties, the infinitesimal change of energy, dE, is a perfect
differential, i.e.,
2
∆E = E(2) − E(1) = ∫ dE (1-3)
1
for the change of energy, ∆E, between the value of E in the final state, “2,” and the initial state,
“1.” This is the mathematical definition of a state function. In contrast, the integral of an arbitrary
mathematical function is dependent on the pathway between initial and final states, and thus the
change in its value from the initial to the final state is not unique.
1-2.2. Work & Heat.

Neither of the two components, work and heat, of the energy defined in eqn (1-2) is a state
function because their changes are dependent on the pathway between states. This can be visualized
by comparing reversible vs. irreversible transitions between initial and final states for various
processes. The reversible transition is ideally characterized by a pathway consisting of small steps
or changes in coordinates. The occurrence of each such step is infinitesimally more probable than
its non-occurrence. In the irreversible transition, each state or sub-state states reached during the
transition is far more probable than that which preceded it. The probability that the reaction can
reverse itself in the latter case is then extremely small and the transition is considered irreversible.
A mechanical example of a reversible and irreversible reaction is provided by a comparison of (i)
the slow reversible, and (ii) sudden irreversible, expansion of a gas, maintained under isothermal
conditions (Fig. 1-1). The reversible expansion may occur through many small consecutive
decreases of pressure (solid arrow pathway in Fig. 1-1A), mediated by a piston (Figure 1-1B). The
irreversible expansion is initiated by a sudden decrease of the external pressure (multi-arrow
pathway, Fig. 1-1A). In the reversible expansion, the system is at equilibrium during the transition,
and it is possible to specify the pressure on, and the volume under, the piston during each small
step, and at any time, and thus to calculate the work done during the compression of the gas. In the
rapid expansion, the pressure on the gas cannot be specified, as it will depend on several parameters
including the magnitude of the pressure decrease and the velocity of the moving piston.

P1
Irreversible
I R Reversible,
Isothermal
path
P2 I
Piston
V1 V2
(A) (B)
Figure 1-1. (A) Pathway of a reversible (R) and irreversible (I) isothermal (constant temperature)
expansion of a gas by: (R) a gradual decrease of pressure from p1 to p2, and associated increase of
volume from V1 to V2. For the I transition, a sudden decrease in the pressure on the piston would
be followed by an increase in volume. (B) Closed chamber with piston mediating expansion and
compression of an enclosed gas.
When the force of the piston is not in equilibrium with the reaction force from the gas
molecules, its effective pressure (p) on the gas is always smaller than in the reversible transition (pI
< pR). Thus, more work will always be done in the reversible transition, and more heat generated in
that which is irreversible. Because the initial and final states of the reversible and irreversible
transitions are the same, the sum of the work done and heat energy generated in the two transitions,
i. e., the total energy change, is the same.
The pressure-volume work accomplished by expansion (or compression) of a gas at

pressure, p, through a volume change, dV, as in the above example, is:
dW = –pdV (1-4)
Note that the convention for pressure-volume work is to carry the negative sign (dW = -pdV); the
result of this sign is that when expansion work is done by the system so that ∆V > 0 and the final
volume is greater than that in the initial state, as in an expansion experiment using the piston of Fig.
1, ∆W and ∆E of the system are negative, and the energy of the system decreases. Conversely,
when compression work is done on the system, ∆V < 0, the final volume is less than the initial, the
work term will be positive, and the energy level of the system the energy of the system is increased.
Then, from eqn (1-1), the First Law of Thermodynamics can be written in terms of the
energy change due to work done on (or by) the system, and the change in its heat energy, as:

dE = dQ – pdV (1-5)
As noted above, for an ideal or dilute gas, the energy, E, is a function only of its temperature, i. e., E
= E(T).
Returning to the isothermal (constant T) expansion experiment shown in Fig. 1-1, the
change in energy, ∆E, between initial and final states = 0 because T is constant. Therefore, the
magnitude of the work done and change in heat energy level are equal, i. e., dQ = pdV = - dW, or
for non-infinitesimal changes, ∆Q = -∆W.
Then, for the reversible pathway in Fig. 1, in which the pressure is decreased slowly and
continuously as the volume increases:
V2 V2 dV V
∆W = − ∫ pdV = − RT ∫ = − RT1n 2 , (1-6)
V1 V1 V V1
where the equation of state (1) was used to substitute for the pressure, p. Because the work done by
the system in the expansion of the gas is negative, the heat change is positive, which means that heat
was added to the system to drive the expansion of the gas.
For the irreversible pathway in Fig. 1-1, again ∆E =0 because the transition of the state
function, energy, occurs between initial (p1, V1, T) and final (p2, V2, T) states that are at the same
temperature. Therefore, again, ∆Q = -∆W. However, unlike the change in the state function, ∆E,
the values of ∆W and ∆Q are different in the irreversible, compared to the reversible, pathway
shown in Fig. 1-1A.
Homework problem 1. (a) Show that the work done by the system in the irreversible transition of
Fig. 1-1 is ∆W = - p 2 (V2 - V1). (b) By comparing the areas under the functions describing the
reversible and irreversible transitions in Fig. 1-1, show that more work is done in the reversible
transition.
Homework problem 2. Calculate the work done by a system such as that shown in Fig. 1-1 for
(a) reversible and (b) irreversible transitions, for each of which ∆E = +20 kJ, but for which ∆Q =
+40 kJ and +30 kJ, respectively.
1-3. Different Kinds of Work.

The different kinds of work, chemical, electrical, and mechanical, respectively, are each a product
of an intensive and extensive variable, i.e., µdn, φdq, and pdV, etc., where µ, φ, and p, chemical
potential, electrical potential, and pressure, are intrinsic variables. The extrinsic variables, n, q, and V,
represent the number of moles of the chemical species and of the electrical charge, respectively, and
V is the volume available for the reaction.
1-4. Enthalpy
The state function, H, called enthalpy, defined as
H ≡ E + pV, (1-7)
is useful because there is a simple expression for the relation between the enthalpy and heat changes
of a reaction under conditions that are common experimentally: i. e., where the only work done is
mechanical, p-V work (i.e., no chemical or electrical work), and the pressure is constant. Under these
conditions, the change in enthalpy, dH, equals the absorption of heat at constant pressure, dQp. That
is,
dH = dQp (1-8)
Homework problem 3. Derive eqn. (1-8) for dH, using the expression for the First Law (eqn 1-
5), substituting dW = -pdV for p-V work, rearranging terms to solve for dQ, and substituting for
dH.

1-5. The Direction of Reactions and Events: The Need for the Second Law
The First Law of Thermodynamics expresses the impossibility of constructing a machine

that can create energy. However, the First Law places no limitations on the possibility of
transforming energy from one form to another, in particular on the conversion of heat energy to
work. The Second Law of Thermodynamics exists because: (i) The First Law does not predict
the direction of reactions arising from thermal motion, particularly the direction relative to the
equilibrium state. (ii) Although heat and mechanical energy are equivalent in their fundamental
nature as forms of energy, there are limitations on the ability to convert heat energy into work. It
is, in fact, impossible to have a physical or chemical process whose only result is: (i) to transform
heat extracted from a source at a single temperature into work, or (ii) to transfer heat from an object
at a given temperature to one at a higher temperature without performing work and consuming work
energy from an external source. The impossibility of the occurrence of these events relates to the
everyday experimental fact that heat energy is transferred spontaneously only down a temperature
gradient, from a higher to lower temperature. This transfer is in the direction toward the state of
thermal equilibrium.
1-5.1. The approach to equilibrium.

At temperatures low enough so that heat and molecular motion can be neglected, the
equilibrium state of a mechanical system is defined by the minimum in its potential energy, φ. φ
will decrease (dφ < 0) until it reaches a minimum (dφ = 0). In all of these processes, energy is
conserved according to the First Law.
However, the First Law of Thermodynamics and the criterion for equilibrium, that potential
energy decreases to a minimum, does not explain many intuitively obvious phenomena that occur in
the presence of heat and thermal motion. For example, it does not explain the ordinary observable
process of evaporation. Considering only the First Law, why should a liquid in an open vessel
evaporate, since the binding forces between liquid molecules are much stronger in the liquid than in
the vapor state? The answer is that evaporation is a result of the statistical tendency of the
molecules to fill all available space. One can calculate that it is extremely unlikely, to the point of an
impossibility, that in the absence of constraining forces, a large number of molecules would remain
confined to a small fraction of the volume available to them.
Thus, one can calculate that the probability that one molecule would be found in one half of
a box (Fig. 1-2) with volume, V, is (1/2V)/V = 1/2. The probability that n molecules would all be
found in that half is (1/2)n. If n is about 1023 for a real box, this event will never be observed. The
most likely state is, intuitively, that in which the particles move freely about the box, and at any given
time are found to be spread evenly about the box, so that the number of distinct rearrangements of
the particles in the box tends toward a maximum. This can be stated in terms of probability. The
Statistical Statement of the Second Law of Thermodynamics is: (i) a system of particles or
molecules that undergoes an irreversible transition, in the absence of external forces, moves
toward a state of greater probability or greater disorder; (ii) In reversible processes, the system
remains in, or very close to, a state of maximum probability
1-6. Entropy and the Second Law of Thermodynamics
The entropy (from Gr., EMBED Equation.3 change, turn) is a state

function whose change in a reaction describes the direction of a reaction due to changes in
heat input or output and the associated molecular rearrangements. There are two ways to
describe the entropy function, S.
1-6.1. In the statistical formulation due to Boltzmann,
S = k ln W , (1-9)

where k = Boltzmann’s constant, which equals R, the gas constant (8.31 J ˚K-1mol-1) divided by
Avogadro’s number, = 1.38 X 10-23 J/°K. That is, Boltzmann's constant is the value of the gas
constant, R, per molecule or atom. In the Boltzmann formulation, W is a probability function
associated with a macroscopic (large) state that consists of many molecules. The value of W in a
given state is determined by the number of arrangements of the molecules of which it is composed,
with each arrangement called a “microstate.” The larger the number of arrangements or
microstates associated with a macrostate, the greater the probability of the macrostate.
Because of the use of the logarithmic function in eqn 8, the product W1 x W2 that describes
the joint occurrence of two different states characterized separately by independent probabilities, W1
and W2, is converted to a sum, ln(W1xW2 ) = ln W1 + ln W2 . This property of additivity, possessed
by state functions such as energy, confers the properties of a state function to the entropy, S, as
defined by Boltzmann (eqn 1-9). Then, the change in the entropy, ∆S, between some final state,
"2", and initial state, "1", is
W
∆S = S(2) – S(1) = k ln 2 (1-9a)
W1
This formulation of the second law in terms of an entropy function that is determined by the relative
probability of molecular distributions emphasizes that irreversible (I) transitions are associated with
increases of the entropy function, and reversilbe (R) transitions with no change of the entropy. That
is,
dSI > 0 (1-10a)

dSR = 0 (1-10b)
1-6.1.1 Example of Calculation of Thermodynamic Probability, W:
Calculate the number of combinations, Wj,k of four identical molecules, N = 4, contained in

a box, on two distinguishable sides, j and k, of the box (Fig. 1-2). The number of independent
arrangements Wj,k resulting from placement of Nj molecules on one side and Nk on the other, with
the two sides of the box having the same volume, each subset with the same number of molecules
weighted equally, and no preferred arrangement on each side of each subset, is:
N!
Wj , k = (1-11)
N j ! Nk !
where N! = N(N – 1)(N – 2) ...(1).
4!
For Nj = 3, Nk = 1, W3,1= = 4, as seen by:
3!1!
A.
bcd acd abd abc
a b c d

4!
The number of rearrangements with Nj = Nk = 2 is W4,2 = = 6, as shown below.
2!2!
B.
cd bd bc ad ac ab
ab ac ad bc bd cd
Figure 1-2. Description of possible arrangements of four identical molecules between two sides of
a box, with (A) 3 molecules on one side and 1 on the other, and (B) 2 on each side. The third
combination would have 4 on one side and none on the other, for which there is only one
4!
arrangement, W 4,0 = = 1. The 4 identical molecules are labeled a, b, c, d for purposes of
4!
discussion.
Thus, the state or combination with the largest number of different arrangements is the one
with equal numbers of molecules on each side of the box. The relative probability of the state
W N/2,N/2 with Nj = Nk becomes increasingly greater with increasing N, and becomes
overwhelmingly more probable compared to arrangements with Nj>>Nk or Nk>>Nj when the
particle number is large or macroscopic, i.e., ca. 1023.
1-6.2. Second Formulation of the Entropy Change; Relation between Heat and
Entropy Change.
The 19th century physicist, Clausius, proposed that the differential entropy change, dS, is
proportional to the heat absorbed,dQrev, for a reversible process, with 1/T [(absolute temperature)-1]
as the constant of proportionality. 1/T is an “integrating factor” for dQ that makes the function,
dQ/T, a perfect differential, as required for a state function. [An integrating factor is a coefficient
for a function that makes it a perfect (i. e., integrable) differential]. It should be emphasized that a
theorem in the mathematics of the integral calculus states that if an integrating factor exists, then it is
unique (Chandrasekhar, 1957).
With dQrev as the heat energy reversibly absorbed by the system, the entropy change, dS,
from Clausius, is:
dQrev
dS = (1-12)
T
and
2
dQrev
∆S = ∫ = S(2) − S(1) (1-12a)
T
1
1-6.2.2. Statement of the Second Law of Thermodynamics Using the Clausius

Formulation of Entropy.
(i) There is a state function called entropy. (ii) The entropy of any isolated system increases
when an irreversible process takes place in the system, dSI > 0 (eqn. 1-10). In an isolated system,
when dQ = 0, the entropy remains constant (and maximum) in all reversible processes, where the
probability of the system configuration remains the same. That is,

dSI > 0, (1-13a)

dSR = 0 (1-13b)
as stated above (Eqns. 1-10a,b) in the Boltzmann formulation.
For irreversible processes in a non-isolated system,
dSI > dQ/T (1-14)
1-6.2.3 Brief Mathematical Summary of Second Law of Thermodynamics
I. Isolated system (dS ≥ 0)

A. Reversible: dSR = 0
B. Irreversible: dSI > 0
dQ
II. Non-Isolated System (dS ≥ )
T
dQ
A. Reversible: dSR =
T
dQ
B. Irreversible: dSI >
T
It can be seen from formulae (1-8) and (1-11 to 1-13) that the units of entropy are cal ˚K-1 or
Joule ˚K-1.
1-6.3. Application of the Second Law to Heat Flow and Temperature Gradients.
Consider an isolated system that consists of two subsystems, “1” and “2.” If an amount
of heat, dQ, is transferred from subsystem 1 at temperature T1 to subsystem 2 at temperature T2,
then “2” absorbs dQ and “1” loses dQ. If there are no other configuration changes in the
system, and the subsystems are at equilibrium, then
dStotal = dS1 + dS2 = -dQ/T1 + dQ/T2 (1-15a)
We know that heat transfer is only possible from system “1” to “2” if T1 > T2. Therefore, from
(1-13) and the temperature inequality,
dStotal > 0, (1-15b)
and heat transfer can only occur from a higher temperature to a lower temperature system, which is
a spontaneous, irreversible process that is readily associated with a positive entropy change.
1-6.4. Relationship between Boltzmann (eqn 1-9) and Clausius (eqn 1-12)
Expressions for the entropy change can again be illustrated by the "molecules in the box"
problem: Consider particles initially confined to one side of a box by a piston (Fig. 1-3A) or a
sliding partition (Fig. 1-3B):

Initial State
A B
Final State
C
Figure 1-3. Two experiments on an isothermal (constant temperature, dE = 0) expansion of a dilute

molecular population in an enclosed vessel. Initial state for (A), a reversible expansion through
absorption of heat, dQ, that exerts a pressure on the piston, performing work, dW = -dQ ≠ 0 on the
system, and dSrev = dQ/T; (B) initial state for irreversible expansion caused by removal of a
partition dividing the chamber, with dW = dQ = dE = 0, and dSirr > 0; (C) the identical final state
that is reached in experiments (A) and (B), with the final volume = twice initial volume. Note: The
expansion of the gas defined by (A, B) could be one phase of the complete cycle of a heat engine,
sometimes called a Carnot cycle.
There are two paths to the final state, in which molecules fill the box, either by pushing the
piston (Fig. 1-3A), or by removing the partition (Fig. 1-3B). The final state achieved through either
path is shown in Fig. 1-3C. The entropy change will be shown below to be ∆S = Rln2 for both
transitions, independent of path as is required for a state function.
1-6.4.1. In the first experiment (Figs.1-3A/C), the change in piston position and molecular
distribution is a result of the absorption of heat. The temperature is held constant by placing the
system in contact with a heat sink. The entropy change from the initial (Fig. 1-3A) to the final (Fig.
1-3C) state is:
dQ
dS = ,
T
for an incremental change of added heat. If the energy, E, is a function only of temperature, using
the First Law, dE = dQ + dW, for constant temperature (dE(T) = 0), then dQ = -dW, and from
eqn. (4),
dW pdV
dS = − =
T T
For 1 mole of a dilute molecular population, from eqn (1-1),
pV=RT

Then, as in the discussion of the reversible transition in Fig. 1-1,

RT dV dV
dS = =R ,
T V V
and
2
dV V
∆S = R∫ = R ln 2 = R ln 2 (1-16a)
1
V V1
since V2=2V1 (Fig. 1-3C).
Homework problem 4. Calculate the entropy change for a reversible expansion of 10 mol of an
ideal gas from an initial volume of 0.1 L to a final volume of 100 L at 27 ° C.
1-6.4.2. In the second experiment, the partition is removed to allow a redistribution of

particles into the entire volume of the box. For a large number of particles (e. g., 1 mole), this
redistribution can never be reversed. If there are N identical molecules, and Nj and Nk in each
compartment, then the relative probability of having a given number Nj and Nk on each side of the
partition is proportional to the number of different ways, Wj,k, in which this may occur, as written
above in eqn (1-10).
N!
Wj , k =
N j ! Nk !
For the problem in Fig. 1-3A, the initial state has all N particles on one side before removal
of the partition. Therefore,
N!
Wj , k = WN , 0 = = W1 ≡ 1
N! 0!
(the symbol “ ≡ ” means, “equivalent by definition”).
After the partition is removed, there are N / 2 particles on each side. Then,
N!
Wj ,k = WN / 2, N / 2 = 2
 N !
2 
From the Boltzmann expression (eqn.1- 7) for S,
W N!
∆S = k ln 2 = k ln 2
W1  N !
2 
since W1 = 1 . One obtains a value of the argument of the logarithm by using "Stirling’s
approximation" for large values of N,
ln N! = N ln N − N
Then, substituting in the expression for ∆S,

 N N N 
∆S = k  N ln N − N − 2 ln −  
  2 2 2 
N
= kN ln = kN ln 2
N
2
the gas constant R ≡ kN (i. e., Boltzmann's const x Avogadro's number, the number of molecules
in a mole, 6.02 x 1023; then for 1 mole,
∆S = R ln 2 (1-16b)
Thus, the entropy change is the same when calculated from “particle in the box”
experiments based on the view of Clausius (section 1-6.4.1; Figs. 1-3A,C) or (ii) that of Boltzmann
(section 1-6.4.2, Figs. 1-3B,C). These experiments described:
(i) a reversible isothermal change involving the input of heat, the performance of work by
the system to drive the piston, a net dE =0, and dSrev = dQ/T;
(ii) an irreversible isothermal change involving a redistribution of particles with dQ =dW
=dE = 0, but dSirr > dQ/T. The conclusion from the above calculations, that ∆S for the two
experiments (pathways) connecting the same initial and final states is the same, supports the
concept that entropy is a state function.
Homework Problem 5. (a)What is the entropy change associated with ice (18 g) melting to water
at 0° C, if the heat of melting is 5.98 kJ/mol. (b) What fractional volume change of this molar
quantity of water vapor would yield the same entropy increase?
6.5. Second Law Implies < 100 % Efficiency of Heat Engines. The simplest heat engine in
which heat can be converted to net work would involve a four step cycle of reversible changes of
pressure, volume, and temperature that is described in the p-V plot (Fig. 1-4) similar to that
described above in Fig. 1-1. Fig. 1-4 describes a cycle made of two isothermal (constant
temperature) transitions, the A→B expansion at temperature T2 and C→D compression at
temperature T1, with T2 > T1. The cycle is completed and the isotherms connected, by the two
adiabatic (∆Q = 0) transitions, the B→C expansion and the D→ A compression.
A
T = T2
1 B
P 4
2
D
3
T = T1 C
V
Figure 1-4. P-V-T plot of of a reversible (Carnot) engine. The direction of the engine cycle, as
described in the text is along p-V coordinates, A → B → C → D. The A-B and C-D curves
describe isotherms at constant temperatures T2 and T1, and the adiabatic (∆Q = 0) transitions, B→
C and D →A, connect the two isotherms.

1-6.5.1. Work Done by System, (I) Isothermal Transitions

In the A→B transition at temperature T2, from eqn. (1-6) the work done by the system
V
∆W2 = − nRT2 ln B (1-17a)
VA
and, since ∆E = 0 at constant T, an amount of heat, ∆Q2, of equal magnitude but opposite sign (for
purposes of energy book-keeping) is absorbed by it in this expansion. Similarly, in the
compressive C→ D transition, work done on the system,
V
∆W1 = − nRT1 ln D (1-17b)
VC
and an equal amount of heat, ∆Q1, is released by it.
1-6.5.2. (II) Adiabatic Transitions, B→C and D→ A: The critical features of the two
adiabatic transitions, B→C and D→A, are: (i) ∆Q = 0, (ii) the work done in the B→C and D→A
transitions is equal and opposite and cancels out because, since ∆Q = 0, ∆EBC = - ∆W BC = - ∆EDA
= ∆WDA; (iii) the ratios of the volumes linking the two adiabatic transitions (Fig. 1-4) are equal,
i. e., VC/VB = VD/VA, and VC/VD = VB/VA (1-18)
Then, the net work done by the system,

V V
∆Wdone = ∆W2 + ∆W1 = − nRT2 ln B − nRT1 ln D
VA VC
VB VC
= − nRT2 ln + nRT1 ln , and from (1-18),
VA VD
V
= − nR ln B (T2 − T1 ) (1-19a)
VA
Because ∆Q2 = − ∆W2 the heat absorbed is (1-17a):
V
∆Qabs = ∆Q2 = nRT2 ln B (1-19b)
VA
The efficiency, ε, of this ideal “Carnot” heat engine is defined as the magnitude of the net work
done, ∆Wdone (formula 1-19a), divided by the heat absorbed, ∆Qabs (formula 1-19b), and
 T 
ε = 1 − 1  ; (1-19c)
 T2 
Note: because T2 > T1, ε < 100 %. This is a result of the work that has to be done on the system at
the lower temperature, T1, to return the engine to its original starting position.
1-6.5.3. The Entropy Change, ∆S, for the Carnot Heat Engine Cycle Equals
Zero. The result (1-19c) for the efficiency < 100 % for the ideal (Carnot) engine could also have
been derived from assuming that the entropy change for the whole cycle must be zero, ∆Scycle = 0,
since it returns to its starting position and entropy is a state function. As an alternative approach, we
will use the information given above for the heat changes in the steps of the cycle to prove that
∆Scycle = 0.
The entropy change for one complete cycle of the heat engine described in Fig. 1-4 is:
∆Scycle = ∆SAB + ∆SBC + ∆SCD + ∆SDA (1-20a)

Because ∆QBC = ∆QDA = 0 for the adiabatic transitions, ∆SBC = ∆SDA = 0, and
∆Scycle = ∆SAB + ∆SCD = ∆QAB / T2 + ∆QCD /T1 . (1-20b)
From the first law (eqn 1-2), and dE = 0 for constant T, the heat absorbed in the A→B isothermic
transition at temperature T2, ∆QAB = - ∆W2 = nRT2 ln(VB / VA ) , and the heat released in the C→ D
isotherm at T1 = ∆QCD = nRT1 ln(VD / VC ) = − nRT1 ln(VB / VA ) . Therefore,
∆Scycle = ∆QAB/ T2 + ∆QCD/T1 = nR ln(VB /VA ) - nR ln(VB /VA ) = 0 (1-20c)
Thus, the heat engine problem demonstrates that the factor (1/T) is unique in its ability to
make a state function out of a function proportional to ∆Q or dQ.
1-7. Free Energy.
The entropy inequality dS ≥ dQrev/T specifies the direction of reactions in which only the
heat change and temperature are specified. Inequalities specifying the direction of chemical
reactions in non-isolated systems are useful. From the first law, dE = dQ + dW, where dW can
include chemical and electrical work as well as that arising from pressure–volume changes.
If dW = dWpV = -pdV for pressure–volume work, then,
dE = dQ - pdV
dQ = dE + pdV
dQp = d ( E + pV ) p
at constant p, where dQp = d(E + pV)p ≡ dH, the heat change at constant pressure (eqn. 1-8).
From the second law, dS ≥ dQ/T, and by substitution,
TdS ≥ dH,
the statement of the 2nd law for a non-isolated system. Rearranging terms,
dH – TdS ≤ 0
Rewriting,
d ( H - TS) p,T ≤ 0
or defining the Gibbs Free Energy, G, is defined as G ≡ H – TS,

dG p,T ≡ dH − TdS (1-21)
The second law of thermodynamics is then reformulated to describe reactions at constant
temperature and pressure:
dG p,T ≤ 0. (1-22)
The reaction is at equilibrium and reversible if
dG p,T = 0 (1-22a)
This is illustrated in a graph of the free energy, G, of an isolated spontaneously reacting
system, e. g., reactant “A” ⇔ product “B”, as a function of the quotient (q = nA/nB) of the
number of moles of A, to those of B (Fig. 1-5). At equilibrium, the free energy, G, is at a minimum
dG
and = 0, i. e., the slope of G as a function of q is zero at the position of the minimum of G, for
dq

small displacements of G in either direction on the q-axis. At all other points on this graph away
from equilibrium, the second law says that the tendency of G will be to decrease, i. e., dG < 0, to a
minimum value.
o
o
Geq dG = 0
dq
(nA / nB)eq
nA / nB
Fig. 1-5. Free energy, G, of the reaction “A” ⇔ “B”, as a function of the quotient, q, of the
molar quantity, nA and nB, of compounds A and B. Note that the equilibrium value of G for the
system, Geq = Gmin. The directions toward equilibrium, marked by arrows, of reactions with G >
Gmin, at coordinates (G, nA/nB) marked by open circles, are shown.
1-7.1. Separation of ∆G into ∆H and T∆S terms. In biochemical and chemical systems
that do not perform any mechanical work, it is useful to consider the nature of the contributions of
the ∆H and T∆S components to ∆G = ∆H -T∆S for reactions in which ∆G ≠ 0. The ∆H term
includes the components of the binding energy and intra- and inter-molecular interactions; ∆S
includes effect of molecular rearrangement. At low temperatures, statistical effects in the
contribution from ∆S will have relatively little weight and the ∆H term will be a major determinant
of the ∆G. However, at high temperatures, the direction of the reaction is dominated by the T∆S
term and the effects of statistical rearrangements, so that the sign of ∆G will tend to be negative.
1-7.1.1. For example, protein denaturation in aqueous solution tends to occur at somewhat
elevated temperatures (≥ 60°C) where the T∆S term that expresses the increase in configurational
space occupied by the protein in the denatured state carries more weight and offsets the large
positive ∆H required to break the hydrogen bonds and favorable hydrophobic interactions that are
responsible for favorable packing of the protein in the native state. Thus, at high temperatures, ∆G
tends to assume more negative values, favoring a high population of the denatured state (Fig. 1-6A).
The “melting” temperature, Tm, of the protein in a calorimetry experiment (Fig. 1-6B), will be
defined by the temperature of the equilibrium, i. e., ∆G = 0, between the native and denatured states,
where there can be a reversible transition between them. Therefore,
∆H
Tm = (1-23)
∆S
For a discussion of the determination of ∆H and ∆S from differential scanning
calorimetry, see V. Bloomfield, Thermodynamics and Statistical Thermodynamics, section 1.7.

(A)
Glu
Lys Arg
Gly Asp Ala

Trp Phe Ile
Asp Glu Phe Phe
Val Ala
His Glu Gly Trp
Ala Leu
Phe Leu
Lys Ala Val
Glu His
Ile
Arg
(B)
Tm(1)
150
130
110
Cp , kJ/mol K
90
70
50 Tm(2)
30
10
-10
0 10 20 30 40 50 60 70 80 90
Temp (o C)
Fig.1-6. Protein denaturation. (A) Schematic diagram of the initial and final final states of a native
→ denatured transition; (B) Endothermic transitions associated with thermal denaturation of the 3
functional domains of colicin E1 modified from (Griko et al., 2000); deconvolution of the major
endotherm into two melting transitions is shown. A representative value of ∆So for protein
denaturation is ~100 cal/mol-˚K.
Homework Problem 6. If the free energy and enthalpy of denaturation of myoglobin are
+13.6 kcal/mol and +42 kcal/mol at 25°C, (a) calculate the associated entropy change; (b) discuss
briefly the reason(s) that the free energy for transfer from the protein core to the bulk aqueous

phase of the side chains of “hydrophobic” amino acids, for example, valine, leucine, and isoleucine
is positive.
1-7.2. Concentration Dependence of the Free Energy.
An expression for the dependence of the free energy, G, on the concentrations of reactants
and products in solution can be obtained by using the identity of the equations of state for ideal
(non-interacting) gases and dilute (non-interacting) solutions. There is an identical dependence of
(i) the free energy of an ideal gas at constant temperature on its partial pressure, dGi = Vdpi, and (ii)
the partial free energy, dG or dµ, of a solute "i" in aqueous solution on its concentration, dG = VdcI.
Because G ≡ H - TS ≡ E + pV – TS, it is clear that G is a function of pressure. Then, from

the differential expression for a reversible non-isolated transition ( dQ = TdS ) involving only p-V
work:
dG = dE + pdV + Vdp –TdS – SdT,
and dE = dQ –pdV; cancelling terms,
dG = Vdp – SdT; p, T not constant.
showing that G is a function of p and T, i. e., G = f(p,T).
In an isothermal (dT = 0) transition, dG = Vdp; (1-24a)

and by analogy with dilute solutions
dG = Vdc (at constant p) (1-24b)
Integration of expression (1-24b) between initial state 1 and final state 2 for n moles yields:
2 2 2
dc c
∆G = ∫ dG = G(2 ) − G(1) = n ∫ Vdc = nRT ∫ = nRT ln 2 (1-25)
1 1 1
c c1
Then, from (1-25), with G(1) = Go and G(2) = G,
{c}
G - G o = nRT ln o , and
{c }
{c}
G = G o + nRTln o = G o + RTln{c}
n
when Co = 1M (1-26)
{ }
c
where G(2) = G, and G(1) = is the standard free energy, co is the concentration under standard
conditions ( co = 1M), and the brackets refer to partial activities in the event that the concentration, c,
is not dilute.
1-7.3. Dependence of ∆G on concentrations of reactants and products.
Considering the reaction,

nAA + nBB →nCC + nDD,
where the coefficients, nA, nB, etc., refer to the number of moles of compounds “A, B, C, and D” that
participate in the reaction, the free energy, GA, of reactant “A” at concentration, cA, is:
∆G o + RTln{ cA }n A ; cAo = 1 M
and combining the free energy expressions for each compound, yields the total free energy change:

Π( products)
∆G = ∆G o + RT ln (1-27a)
Π(reac tan ts)
{ c }nC { c }n D
∆G = ∆G o + RT ln C n A D n B (1-27b)
{ c A } { cB }
where ∆G = (GC + GD ) – (GA + G B), and ∆Go = (G C o+ G Do) – (G A o+ GBo), and the quantities,
cA, cB, etc., in curly brackets are the concentrations or activities of compounds A, B, C, and D. At
equilibrium, ∆G = 0, and therefore
o {cC}nC {cD}nD 
∆G = − RT ln  n n  ,
 {c A} A {cB} B  eq
where the subscript, “eq,” refers to evaluation of the concentrations or activities at equilibrium.
The quotient of products to reactants evaluated at equilibrium is defined as the equilibrium constant
Keq Then,
∆G o = − RT ln Keq = −2.3 RT log Keq ; ∆Go < 0 when Keq > 1 (1-28)
and ∆Go > 0 when Keq < 1.
1-7.4. Using ∆G to predict reaction direction near its mid-point

o
∆G o is the free energy change for conversion of a standard amount of reactant [1 molar for
solutes, 1 atmosphere of pressure [1.013 x 105 Pascal (Pa)] for gases, at 25oC] to the same standard
amount of product. The complete conversion of a standard amount of reactant to product may be
regarded as involving (i) a conversion of the reactant to an equilibrium mixture (with negative ∆G
because the reaction goes toward equilibrium), and then (ii) conversion of the equilibrium mixture
to products (with a + ∆G since the reaction is going away from equilibrium). If Keq > 1, then the
negative ∆G step outweighs that with positive ∆G.
In addition, the sign of ∆G o can indicate the direction of the reaction at or near its midpoint,
where the concentration of products, “p”, equals that of the reactants, “r” [i.e., [πi(pi)/ [πi(ri)] = 1,
1n [πi(pi)/ [πi(ri)] = 1n 1 = 0, and ∆G = ∆G o in Eq. (1-20)]. However, when the activities of
reactants and products are not equal, it is the sign of ∆G, and not that of ∆G o that
determines the direction of the reaction.
Homework problem 7. (a) Applying the relation, ∆G0 =–2.3RT l og10Keq, to acid-base reactions,
AH → A– + H+, for which the pK = –log10Keq, and ∆G o = 2.3 RT • (pK). What is the difference in
the ∆Go at 25oC for an acid with a pK of (i) 4 and (ii) 7? (b) What is the change in ∆G0 of a
reaction that accompanies a 100-fold increase in Keq at 25oC? (c) What is the effect on the Keq of a
decrease of 1.36 kcal/mol in the ∆G0 at the same temperature?
Homework problem 8. Given the reaction, r(reactant) ↔ p(product) proceeding at 25°C with a
∆Go = -1.36 kcal/mol for the direction r → p, in what direction does the reaction tend to proceed
when it operates between concentrations or activities of {r} and {p} that are: (a) 0. 91 and 9.1 µM;
(b) 5 and 5 µM; (c) 9.1 and 0.91 µM; (d) 9.91 and 0.09 µM?

Homework problem 9. The hydrophobic nature of amino acids has been determined from the
following kind of experiment: The ∆Ho and ∆Go for the transfer of pure benzene to H2O at 18°C is
0 and 19.4 kJ/mol. What is the ∆So for this reaction? Explain the sign of the ∆So.
1-7.5 Additivity or Lack of Additivity of ∆G, ∆H, and ∆S.

Thermodynamic additivity of changes in free energy, enthalpy, and entropy is implicit in the
nature of these quantities as state functions. However, a requirement of additivity is that the
processes for which ∆G, ∆H, or ∆S are being added must be independent. Thus, if two
components, A and B, contribute to a net process, the free energy change, ∆G = ∆GA + ∆GB, is a
valid expression only if components A and B are independent (Dill, 1997). It is, in fact,
experimentally difficult to establish this requirement. One example cited by (Dill, 1997) is the
transfer free energy, ∆Gg, of glycine from oil to water. It has been found that ∆Gg = 1.270 or
0.895 kcal/mol if ∆Gg is calculated, respectively, as the difference in the transfer ∆G of (i) the
tripeptide minus the dipeptide, or (ii) the dipeptide minus glycine. The explanation of this
difference is that the environment around the different Gly peptides is variable. Such experimental
variability, in homogeneity, sometimes arising from conformational change, implies that additivity
of thermodynamic state function parameters cannot be taken for granted.
1-8. Other Kinds of Work
The free energy change, dG, in a reversible transition at constant (p,T) is equal to the work
done exclusive of p-V work. Other kinds of work commonly encountered in biochemistry and
biophysics are chemical, electrical, and electrochemical, denoted by dWce. It can be shown by
substituting (dW –pdV) into the expression for the First Law,
dE = dQ + dW = dQ + (dWce –pdV)
into the expression for
dG = d(H-TS)]p,T = [dE + pdV + Vdp –TdS –SdT]p,T = dQ + (dWc,e–pdV) + pdV –TdS, that
dGp,T = dWce . (1-29)
When it is specified that this work is done in a reversible transition, the work done is the
maximum that can be done under the conditions of this transition. As discussed above, when dW is
negative and positive, respectively, work is done by or on the system and the free energy of the
system will correspondingly decrease or increase.
A logical extension of the equality/inequality relationships discussed above (Eqns. 1-22, 1-
22a) is that ∆G = ∆W ce = 0 at equilibrium for dW ce work. The physical-chemical formalism for
dWce also requires that dG < 0 for irreversible or spontaneous reactions.
1-8.1. Electrical, Chemical Work
Electrical work is carried out by moving a charge (units, coulombs, "C") across a potential,
∆E or ∆ψ, in volts). The charge is equal to the Faraday constant (molar value of the electronic
charge (9.6487 x 104 coulombs/mole, or 23.06 kcal mol-1 V-1)), multiplied by the valence of the
charge, z, the sign of the charge, e (±1), and the number, n, of moles transferred. Then,
∆Gelec = ∆Welec = ni zeF • ∆E (1-30)

where ze is the charge on the ionic species, and ∆E is the electrical, or oxidation-reduction potential
difference, in volts. The other common mode of electrical energy storage is in the trans–membrane
electrical potential difference, ∆ψ, which is typically approximately 100 mV. Then, for electrical
work, the free energy level, Gelec, corresponding to a potential E or ψ, is,
Gelec = ni zeF • E (1-31a)
and, Gelec = ni zeF • Ψ (1-31b)
The differential free energy change corresponding to a small change, dni, in ni at an
electrical potential of E or ψ, is:
dGelec = dni zeF • E (1-32a)

and, dGelec = dni zeF • Ψ (1-32b)
1-8.2. Chemical Work and Chemical Potential
The chemical potential, µi, of compound i is the free energy per mole,
 ∂G 
 ∂n  ≡ µi (1-33)
 i
T , p,n ≠ n
j i
The symbol ∂ stands for partial derivative, the derivative with respect to ni with T, p, with the other
n j ≠ ni held constant.
O
i
I j
Figure 1-7. Transport of solutes i, j, k from the outside compartment O to the inside, I.
Problems concerning the movement of solutes, dni, across membrane boundaries define the
field of transport. When the solute is accumulated against its concentration gradient, one speaks
of active transport because this process requires energy. The chemical potential, µ, is an
important parameter for transport problems because it represents the ∆G per mole of component
transported in or out of the membrane-enclosed system.
Consider a two-component system [outside, O; inside (e.g., cytoplasm), I]. Then, the free
energy, G = G I + GO . If dni, dnj, dnk moles of components i, j, and k are transferred from the
solution outside, O, to the inside of the cell, I (Fig. 1-7), then

dn I = dni = moles of i gained by phase I, (1-34a)

dn j = dn j = moles of j gained by phase I, etc.
I
dniO = − dni = moles of i lost by phase O, (1-34b)

dn j = − dn j = moles of j lost by phase O, etc.
O
The change in free energy of the system, dGi,chem for the chemical work done on it through
movement of only dni is:
dGi , chem = dG O + dG I (1-35)

O I
 ∂G   ∂G 
=   ⋅ dni O +   ⋅ dni I
 ∂ni   ∂ni 
 ∂G  I  ∂G  O 
=   −    dni , (1-36a)
 ∂ni   ∂ni  
From Eqns. 1-33a, b above,
dGi = ( µ i I − µi O ) ⋅ dni ,
from the definition given above for chemical potential. Thus,

dGi( chem ) = ∑ µi ⋅ dni , (1-36b)
where the chemical potential of component i is summed (∑ ) over the compartments O and I.
For net transport, µ ≠ µ . The reaction proceeds then in the “forward” (inward) direction
O
i
I
i
if µ > µiI . This corresponds to the concentration of component “I” being greater in the outside
O
i
compared to the inside component. Therefore, this spontaneous flow of solute from higher to lower
concentration, like that of heat from higher to lower temperature is characterized by dG < 0.
At equilibrium, µiO = µiI and dGi = 0 .
In general, the free energy change arising from movement of dni at chemical potential µi is:
dGi = µi dni (1-36c)
The combined free energy change for electrical (Eqns. 1-32a,b) and chemical work (Eqn. 1-36c)
arising from movement of dni from chemical potential µi and electrical potential, E (or Ψ) is
dGi = dni zeF • E + µi dni (1-37)
Dropping the subscript i, the sum of the electrical (e) and chemical (c) terms in the free energy is the
electro-chemical (ec) free energy dGec .
dGec = µ • dn + zeFE • dn = µ̃ • dn (1-38)
where the electrochemical potential, µ˜ = µ + zeF • E = dGec / dn .
The significance of dGec is that the accumulation or escaping tendency of an ion is
determined by both chemical and electrical potential gradients.
1-8.3 Electrochemical Work; The Electrochemical Potential.

The dependence of the chemical potential, µ, on concentration has the same form as does the
free energy, G (Eqn. 1-26):
{c} {c}
µ = µ o + RTln = µ o + 2.3 RTlog10 (1-39)
{c }
o
{c }
o
where µo, co are the chemical potential and concentration (1M) under standard conditions.
Then, from (1-37), the complete expression for the electrochemical potential of a charged
solute is
{c}
µ̃ = µ o + RTln o + zeF • ( E − E o ) , (1-40)
{c}
where Eo is the electrical potential in the standard state.
As the difference in chemical potential is a measure of the escaping tendency of uncharged
molecules due to a concentration gradient, the electrochemical potential describes this tendency of
charged molecules due to both a concentration gradient and an electrical potential. When applied to
H+ or Na+ gradients across energy transducing membranes, Eqn 1-38 underlies the chemiosmotic
framework for energy coupling (Mitchell, 1966)
1-8.4. Thermodynamics of Ion Gradients
Consider the application of the electrochemical potential (eqn 1-40) to proton (H+)
translocation driven by a membrane potential , with the protonic charge, ze = +1,
µ˜ = µ o + RT • ln{H +} + F • Ψ (1-41)
omitting the standard concentration, co, and standard or reference potential then,
µ˜ = µ o + 2.3 RT • log10 {H +} + F • Ψ (1-42)
µ˜ = µ o − 2.3 RT • pH + F • Ψ (1-42a)
since pH ≡ - log {H+}.
The gradient of the electrochemical potential for protons, ∆µ̃ H + , from one side
of a membrane, for example, initial state, “outside” ≡ “O” to the final, “inside,” ≡ “I”, is:
H I+
∆µ̃ H [(Final – Initial)]= RT • ln + + F( ΨI − ΨO ) , in kJ/mol, or
+ (1-43)
HO
H I+
∆µ̃ H = 2.3 RT • log10
+ + F( ΨI − ΨO ) (1-43a)
HO+
= 5.69 • ( pH0 − pHI ) + F( ΨI − ΨO ) at 25 °C, (1-43b)

where 2.3RT = 5.69 kJ/mol at 25°C, and (-log H+) = pH. Then,
∆µ˜ H + = F ⋅ ∆ψ − 2.3 RT ⋅ ∆pH (1-44)
where ∆Ψ = ΨI − ΨO and ∆pH = pHI –pHO.

Eqn (1-44) is the fundamental formula that describes energy transduction in energy-
transducing proton-translocating membrane proteins in the ‘chemiosmotic” framework (Mitchell,

1966). In this framework, energy is stored in (i) the pH gradient that is generated by electron
transport or ATP hydrolysis, coupled to the pumping of protons across the membrane, and (ii) the
trans-membrane electrical potential arising from electrically uncompensated transfer of electrons or
protons across the membrane. It is assumed in Eqn (1-44) that the membrane is impermeable to the
movement of protons except through pathways mediated by the energy transducing membrane
proteins (i. e., electron transport, ATP synthase/ATPase, transport proteins).
Some organisms may alternatively utilize sodium to generate the trans-membrane
electrochemical potential. Thus, for an analogous electrochemical gradient involving the
translocation of Na+ from its initial, “I”, to its final state, “F,”
(Na + ) F
∆µ˜ Na + = F ⋅ ∆ψ + 2.3 RT ⋅ log10 , (1-45)
(Na + ) I
with ∆ψ = ( ΨF − ψ I ).
1-8.4.1. Proton-Motive Force.
Using the same convention and notation for defining ∆ψ and ∆pH that form the basis for eqns
(1-44, 1-45), the term “proton-motive force”, ∆p, the “pmf”, is defined as
∆µ˜ H + 2.3 RT
∆p = = ∆Ψ − ⋅ ∆pH, (1-46)
F F
with units of electrical potential, in volts or millivolts; substituting 59 mV for 2.3 RT/F evaluated
at 25°C.
∆p = ∆Ψ − 59 ⋅ ∆pH in millivolts (1-46a)
The utility of formulation (1-46) for chemiosmotic energy transduction is that it makes clear that
every change of one pH unit in the ∆pH is equivalent to a 59 mV change in the membrane potential,
∆Ψ. On the other hand, it should be noted that the term “pmf” is a bioenergetics jargon
incorrectly describes the dimensionality of expressions (1-44, 1-45).
1-8.5. Formation of ∆µ̃ H + Across the Inner Membranes of Bacteria and

Mitochondria, and Chloroplast Thylakoid Membranes
1. Chloroplasts (Fig. 1-8A). The ∆µH+ formed by H+ movement driven by photosynthetic electron
transport from the chloroplast stroma on the outside of the membrane (initial state) to the internal
compartment of the lumen (final state), so that the designation of initial and final states for the
formation of the ∆µ̃ H + is:
∆µ˜ H + = F∆ψ − 2.3 RT ⋅ ∆pH (1-47)
∆ψ = ψ in − ψ out ,
∆pH = pH in − pH out ,
and ∆µ˜ H + = ( µ˜ H + )in − ( µ˜ H + )out

ADP + Pi
ATP
µ(H+)p µ(H+)n µ(H+)p

µ(H+)n
H+ ADP + Pi
ATP
ETC ETC
e- e-
H+
(A) (B)
Figure 1-8. Vectorial proton pumping by the electron transport chain (“out” → “in”) in (A)
chloroplast thylakoid, sub-mitochondrial, and chromatophore (sub- photosynthetic bacterial)
membranes; (B)“in” → “out” proton transfer in intact mitochondria and bacterial cells. H+ flow
through ATP synthase is also shown. ETC, electron transfer chain
2. Bacterial Cells and Mitochondria. In contrast to chloroplast thylakoid membranes, H+

movement occurs from “in” to “out” in the energization of the inner mitochondrial or bacterial
cytoplasmic membrane by electron transport or ATP hydrolysis. Then, it is important to keep in
mind that the ∆µ̃ H + associated with this energization, referenced to the difference between final and
initial states, “out” minus “in” has a sign opposite to those in eqns (1-44,47). That is, as in (1-
47),
∆µ˜ H + = F∆ψ − 2.3 RT ⋅ ∆pH ; however,
∆ψ = ψ out − ψ in , (1-48)
∆pH = pH out − pH in ,
and ∆µ˜ H + = ( µ H + )out − ( µ H + )in , for the differences between initial and final states.
1-8.5.3. Comments on the Impermeability of Biological Membranes to Ions; the

Born Energy.
It is implicit in figures 8A,B that the only pathways for protons to cross the membrane (a) to
generate the trans-membrane potential or pH gradient, and (b) to utilize the electrochemical gradient
for the synthesis of ATP, or the accumulation of solutes in active transport (see below, Fig. 9), are
through specific integral membrane proteins. As a consequence of the apolar nature of the lipid acyl
chain core of membranes, the lipid part of the membrane is essentially impermeable to the

penetration and translocation of charged ions. The apolar nature of membranes can also be
described in terms of the low value of the dielectric constant (ε = 2) of the acyl chains. The
dielectric constant, denoted by ε, of a medium is a measure of its electrical polarizability. As a result
of the low dielectric constant, an ion that is inserted into the bilayer will find no counter charges to
balance it. In the absence of counter charges, the ion will thus generate a high electric field intensity
and electric field energy density inside the apolar medium. Thus, the difficulty in inserting an ion
into a “greasy” medium of low dielectric constant is explained by the relative absence of counter
charges or of the barrier created by the necessary generation of a high internal electric field energy.
The barrier is called the “Born energy”, after the physicist, Max Born, who described it. The
energy barrier or Born energy, Eb, the difference in the energy of the ion electric field in the
membrane and in water, is:
z2  1 1 
Eb = 695 − , in kJ/mol,
r  ε m ε H2 O 
where z and r are the valence and radius (Å) of the ion. For the fully charged group of the Arg,
Asp, and Glu ion, z2 and r are 1 and 2.5 Å, respectively (Krishtalik and Cramer, 1995). εm and εH2O
are the dielectric constants of water and the lipid bilayer, which are 78 and approximately 2,
respectively. The energy barrier can be calculated from the above formula for the Born energy to be
135 kJ/mol = 32 kcal/mol. This barrier can be thought of as an activation energy, ∆G‡, for
translocation of a single charge. From reaction rate theory, the effect of this ∆G‡ on the rate of
−135
−∆G‡
proton translocation is then e = e 2.48 = e −55 = 10-24. This implies that the rate at which a
RT
single charge will penetrate a lipid bilayer membrane is very small, and the lipid bilayer membrane
can be thought of as impermeable to single charges including protons.
8.5.4 How is the Membrane Potential Generated?
The generation of the membrane potential and pH gradient by the movement of the
positively charged proton across the membrane is illustrated in Figs. 8A,B. If we know the relation

between electrical charge moved across the membrane per unit area, and the resulting electrical
potential, we can know the number of protons, ions, or electrons that must move across this area to
generate a physiologically meaningful membrane potential.
The physical relation between charge (Q) moved and the potential ∆Ψ is a constant of
proportionality called the capacitance. Thus, Q = C x ∆Ψ , or ∆Ψ = Q/C. Thus, the larger the
capacitance, the more charge has to be moved across the membrane in order to generate a given
value of ∆Ψ. For a planar electrical capacitor that can be viewed as a small segment of a biological
membrane (Figure 8C), the specific capacitance, i. e., the capacitance per unit area, is proportional to
the dielectric constant, ε, and inversely proportional to the distance, d, between the two sides of the
capacitor. That is, C per unit area = ε/d. Because the distance between the two sides of the biological
membrane is small, approximately 35 Å, the specific capacitance of such membranes is large, ca. 1
µFarad per cm2, even though the dielectric constant is small.
∆Ψ
+Q -Q
ε = 78
ε= 2
→35 Å←
Figure 1-8C. A simple planar capacitor as a model for a local segment of a biological lipid bilayer
membrane.
Relation between ∆Ψand charge transfer across a membrane. The ability of membranes
to sustain an energetically significant ∆Ψas a result of the movement of a small number of
electrons or ions across the membrane is a consequence of the large value of the specific membrane
capacitance. The difference in the charge, Q, translocated to one side from the other results in a
potential ( ∆Ψ) across the dielectric. With

Q = C x ∆Ψ
and C = 1 µFarad/cm2, if the potential across the membrane is ∆Ψ= 100 mV, the number of
transferred charges is approximately 1 per 104 Å2, i. e., approximately (a) 1 charge per membrane
protein complex subtending an area (100 Å x 100 Å) of this size, or (b) per 160 lipid molecules in
the membrane monolayer if the area subtended per lipid molecule is 60 Å2.
1-9. Thermodynamics of ∆µ̃ H + -Linked Active Processes

1-9.1. Active transport; Symport
anion- H+ cation+ H+
H+ H+
- -
OH-
- Na+
OH-
- - OH- -
- anion- - S -
S H+ H+
A B C
Fig. 1-9. Diagramatic representation of a neutral or charged solute into a cell by symport (A) or
antiport (C) mechanisms, and of a charged solute S+, e. g., K+, by a uniport mechanism (B).
Modified from (Cramer and Knaff, 1991).
The uptake of solute S+z (charge z) from an initial state, “out,” in the extracellular milieu
outside the cell to a final state, “in,” when the solute has been transported into the cytoplasm, is
accompanied by the uptake (symport) of n protons (Fig. 1-9A), that is driven by a proton flux
down the gradient of the proton electrochemical potential, ∆µ̃ H + from the side of the positive to that
of negative µ̃ H + . The uptake results in a ∆µ̃ S + Z for substrate accumulation. If all of the ∆µ̃ H +
generated by electron transport and ATP hydrolysis is utilized by proton uptake into the cell
associated in the active transport process, then this free energy change, n∆µ̃ H + , equals the ∆µ̃ S + Z
per mole of solute, S+z, that is accumulated, from an initial extracellular state to a final cytoplasmic
compartment:

∆G + = n∆µ˜ H + + ∆µ˜ S = 0 , (1-49)

SZ
where n is the number of moles of H+ that would have to move down the ∆µ̃ H + gradient from
outside to inside to generate the accumulation. Note that ∆µ̃ S is positive for active transport for
which the solute accumulation ratio is > 1, and ∆µ̃ H + for the utilization of the electrochemical
potential initially generated by electron transport and ATP hydrolysis must then be negative in eqn
(1-49). Then, for the utilization of the ∆µ̃ H + as described in Fig. 1-9A,
∆µ˜ H + = F∆Ψ − 2.3 RT∆pH (1-50)
where ∆pH = pHI − pHO and ∆Ψ = ΨI − ΨO .
By analogy with eqns (1-43a,1-44),

{SI + z}
∆µ˜ S = 2.3 RT • log10 + zF • ∆ψ (1-51)
{SO + z}
Substituting (1-50) and (1-51) into (1-49),
{SI + z}
2.3 RT • log10 + zF • ∆ψ + n[ F∆Ψ − 2.3 RT • ∆pH ] = 0,
{SO + z}
{SI + z}
dividing by 2.3RT, and solving for the accumulation ratio, log10 ,
{SO + z}
Si + z ∆ψ
log10 +z
= n∆pH − (n + z ) ⋅ (1-52)
S0 Z
with 2.3RT/F = 59 mV ≡ Z at 25°C, expressed in millivolts. This is the expression for accumulation
of solute S with charge z by a symport mechanism. The solute uses n protons (n ⋅ H + ) per solute
molecule, with the transport driven by a trans-membrane pH gradient (∆pH) and membrane
potential (∆ψ).
Lactose is an example of a solute whose accumulation is driven by a symport
mechanism (Ramos and Kaback, 1977) for which n = 1 (West and Mitchell, 1972) for transport by
the lactose permease.
1-9.2 Special Case; Uniport.
For n = 0, The mechanism is called uniport (Fig. 1-9B), is driven only by ∆ψ because the
proton flux does not participate in the transport mechanism. Setting n= 0 in eqn (1-52) leads to:
S +z ∆ψ
log10 I + z = − z ⋅ (1-53)
SO Z
This is the formula that describes accumulation of solute through active transport by a Uniport
mechanism. To achieve an accumulation ratio of 100 for a singly charged species, a ∆Ψ = ΨI − ΨO
of -118 mV would be required at 25°C. Note that negative sign for ΨI − ΨO implies that the
potential is negative on the inside of the membrane relative to the outside or external side. This is
the sign of the trans-membrane electrical potential in almost all biological membranes, including the
bacterial cytoplasmic membrane and the mitochondrial inner membrane, which are discussed below.

One major exception is the chloroplast thylakoid membrane (also, see below).
1-9.3. Antiport mechanism
For an antiport transport (e. g., Na+ - H+ exchange) as described in Fig.1-9C, in contrast to
symport and uniport (Figs. 1-9A,B) the initial and final states for solute movement are, respectively,
the inside, “I” and outside “O”, of the membrane. The initial and final states for proton transfer
and utilization of the energy stored in the proton electrochemical gradient are independent of the
transport mechanism and are the same for the antiport as for the symport and uniport mechanisms.
Then,
S +z
∆µ˜ S + z = 2.3 RT ⋅ log10 o+ z − zF ⋅ ∆ψ , (1-54)
Si
with signs reversed from Eqn. (1-51) because of reversal of initial and final states for solute
transport, and ∆ψ = ψ in − ψ out .
Using ∆µ̃H + as in the symport case above, with ‘n’protons utilized per solute molecule
S +z
transported, combining ∆µ̃H + and ∆µ̃ S + z , and solving for log10 i + z ,
So
S +z ∆ψ
log10 i + z = (n − z ) ⋅ − n ⋅ ∆pH. (1-55)
So Z
This is the formula that describes the accumulation of solute through an antiport mechanism. If n
= z, then the charge movement would be neutral, ∆ψ has no effect, and
S +z
log10 i + z = − n ⋅ ∆pH. (1-56)
So
1-9.4. A mechanistic problem arises when the mechanism of proton translocation through the
transport protein that is embedded in the membrane bilayer is considered. Carboxylate residues
have been shown to be intraprotein and intramembrane H+ carriers. Whereas the pK of Asp in
solution is nominally about 4, the pK in the membrane environment is commonly 7-9. This reflects
the energetic tendency of the residue to retain the neutral protonated, as opposed to the charged
unprotonated, form in the low dielectric membrane. The pK of 7-9 facilitates H+ transfer reactions
in the membrane. However, to achieve such altered pK values, energy would be required in the
assembly and biogenesis of the membrane protein.
Homework problem 10. Calculate the ∆G at 25 C for the protonation and neutralization at pH 7
of half the amount of aspartic acid (A- + H+ →AH) in a membrane protein, if the effective pK of
this residue in the membrane is (a) 4.0, and (b) 8.0. It may be helpful to derive the formula ∆G = -
1.36 (pK-pH).
Homework problem 11. (a) What is the accumulation ratio of a monovalent anionic species
transported by E. coli through a proton symport mechanism, if ∆Ψ= -118 mV and ∆pH = +1? (b)
What is the ratio of accumulation under the same conditions of an uncharged sugar, e. g., lactose?
The number of protons, n, translocated in (a) and (b) = 1. (c) Consider transport of Cl- into inside-
out membranes, ∆Ψpositive inside. (i) Derive an expression for the log of the accumulation ratio

according to a uniport mechanism. By how much does the ratio change when ∆Ψis increased from
+ 59 to + 118 mV?
1-10. Thermodynamics of ∆µ̃H + -Linked ATP Synthesis

The synthesis of ATP coupled to membrane energy storage is also linked to the utilization
of the proton electrochemical gradient across the membrane, again from the side of positive (p)
µ̃ H + , to the side that has a µ̃ H + that is more negative (n). The “p” side is in the inside or lumen
side of the chloroplast thylakoid membrane (Fig. 1-8A), and the outside of the mitochondrial inner
membrane or bacterial cytoplasmic membrane (Fig 1-8B). Thus, synthesis of ATP from ADP and
orthophosphate that is coupled to a proton flux through the ∆µ̃H + , with n protons required per mole
of ATP synthesized, can be written:
ADP + Pi + nH p + → ATP + H2O + nHn + , (1-57)
for which the free energy for ATP synthesis is:
{ ATP}
∆GATP = ∆GATP
o
+ 2.3 RT log10 , (1-57a)
{ ADP}{Pi }
o
with ∆GATP for synthesis of ATP is ≈ 8 kcal/mol = 33.5 kJ/mol (pH 8, Mg+2).
If all of the free energy stored in the proton electrochemical potential is utilized ( ∆µ˜ H + < 0) for
ATP synthesis, the total free energy change would be zero. Thus,
∆Gtot = n ⋅ ∆µ̃H + + ∆GATP = 0 (1-58)
substituting for ∆GATP and using (1-57a),
{ATP}
− n ⋅ ∆µ˜ H + = ∆G o + 2.3 RT ⋅ log10 ; (1-59)
{ADP}{Pi}
then,
{ ATP} −1
log10 = ( ∆G o + n ⋅ ∆µ˜ H ) (1-60)
{ ADP}{Pi } 2.3 RT
+
1-10.1 ATP Synthesis in Chloroplast Thylakoid, Sub-Mitochondrial and

Chromatophore Membranes.
A positive ( ∆µ̃ H + ) is generated in the chloroplast thylakoid lumen from protons pumped
p
into the internal space by the photosynthetic electron transport chain or ATP hydrolysis. The
discharge of this positive potential from the “inside” (initial state) to the “outside” (final state) is
coupled to the synthesis of ATP by the ATP synthase (mushroom-shaped structure in Fig 1-8).
These membranes have a topology, with respect to the direction of proton pumping and the
orientation of the ATP synthase that is the opposite of right side-out bacterial and mitochondrial
membranes. Considering the energy balance, as in (1-58) and with
∆ψ = ψ O − ψ I ; ∆pH = pHO − pH I , one obtains the expression,
{ATP} −1
log10 = ( ∆G o + n ⋅ ∆µ˜ H + ) (1-61)
{ADP}{Pi} 2.3 RT

with ∆µ H + = F ⋅ ∆ψ − 2.3 RT ⋅ ∆pH , from Eqn (1-44).
Using the parameters that are typical for the energized thylakoid membrane, one may estimate the
poise (ratio) of ATP/ADP: ∆ψ = 0 mV, ∆pH = pH final - pHinitial = pHout - pHin = + 2.5, and from
the latest data on the proton:ATP stoichiometry in ATP synthesis, n = 3-4. If ∆GATP
o
is 8 kcal/mol
and the orthophosphate concentration (Pi) is 10 mM, and n = 4,
{ ATP } −1 −1
log10 = ( ∆G o + n ⋅ ∆µ˜ H + ) = [ +8 + n( F ⋅ ∆Ψ − 2.3 RT ⋅ ∆pH )]
{ADP}{Pi} 2.3 RT 2.3 RT
= − − n ⋅ ∆pH  = −(5.9 − 10) = +4.1
{ATP} 8
log10
{ADP}{Pi}  1.36 
Substituting {Pi} = 0.01 M,
{ATP}
log10 = 2.1 , the ratio of concentration of ATP to ADP (ATP:ADP) = 126.
{ADP}
1-10.2 ATP Synthesis in Mitochondria and Bacteria.

A positive ( µ˜ H + ) p in the periplasmic (bacteria) or inter-membrane (mitochondria) space is
generated by H+ pumped out of the membrane by the electron transfer chain or the ATPase. This
also generates a relatively negative ( µ˜ H + )n in the cytoplasm or mitochondrial matrix (internal)
space. Energy requiring synthesis of ATP from ADP and orthophosphate again results from the
downhill discharge of the ∆µ̃H + through the ATP synthase enzyme in which the proton flux is
utilized enzymatically. In this case, for ATP synthesis, the initial and final states are “outside” (O)
and “inside” (I), respectively (Fig 1-8B).
o
Example: Calculate the {ATP}:{ADP}{Pi} poise with ∆GATP = +8 kcal/mol (pH 8), for
physiological values of ∆Ψ and ∆pH, e.g., ∆ψ = -118 mV = ψ in − ψ out ; ∆pH = + 0.5 =
pHin − pHout ; assume that the number of H+ that pass through the ATP synthase per molecule of
ATP synthesized is 4 (n =4 ). These values for ∆Ψ and ∆pH are typical for bacterial and
mitochondrial membranes. From (1-60),
{ ATP} −1 −1
log10 =
{ ADP}{Pi} 2.3 RT
( )
∆G o + n ⋅ ∆µ˜ H + =
2.3 RT
[ +8 + n( F ⋅ ∆Ψ − 2.3 RT ⋅ ∆pH )]
∆Ψ −118
= −  ) − n ⋅ ∆pH  = − 5.9 + n − 0.5n, where Z = 2.3 RT / F
8
+ n(
1.36 Z   59 
= −[5.9 – 2(4) – (0.5)4] = - (5.9 - 10) = + 4.1

If {Pi} = 10 mM, then {ATP}:{ADP} = 126. The identical results obtained in the
calculation of Section (1-10.1) for ∆pH = 2.5, and in (10.2) for ∆Ψ = -118 mV and ∆pH = +0.5
illustrate that these two sets of conditions generate the same ∆µ̃ H + (Eqn. 1-44) or proton-motive
force (Eqn. 1-46). That is, the ∆pH and ∆Ψ are thermodynamically exchangeable in the energy
transduction of biological membranes.
Homework problem 12. (a) Solve problem 11a for z = 1, n = 1, ∆Ψ = -177 mV, ∆pH = 0.

(b) Solve problem 11b for z = 0, n = 1, ∆Ψ = -59mV, ∆pH = 2.
Homework problem 13. What is the concentration of Na+ ion in the E. coli cytoplasm if it is
transported from the cytoplasm via a Na+ - H+ (n = 1) antiport exchange mechanism, the Na+
concentration in the periplasm = 100 mM, and ∆pH = + 1?
Homework problem 14. Calculate the value of the membrane potential, ∆ψ, needed to drive ATP
synthesis to an effective equilibrium (∆G = 0) if the required ∆GATP for ATP synthesis, ∆GATP =
+12.25 kcal/mol in (i) chloroplasts, (ii) mitochondria, if the ∆pH associated with ATP synthesis is
+3 and +1, respectively. Assume that the number of protons required for synthesis of one ATP
molecule by the ATP synthase = 4.
Homework problem 15. (a) What value of the ∆pH is needed in chloroplast thylakoids to sustain
an {ATP}/{ADP}{Pi} poise of 1000 if ∆Ψ= 0, and n = 4?; (b) what value of the ∆Ψin
mitochondria if the ∆pH = 0? ∆GoATP = +8 kcal/mol for (a) and (b).
Homework problem 16. Calculate the ATP/ADP ratio that can be obtained from the ∆µ̃ H + = + 4
kcal/mol that has been generated by an electron transport chain and whose complete discharge will
release –4 kcal/mol for coupling to ATP synthesis. n(ATP) = 4 (a) for the mitochondrial or
chloroplast ATP synthase, and (b) n(ATP) = 1 for the vacuolar ATPase of the mold, Neurospora
crassa. Assume ∆G o ATP = + 8 kcal/mol, and {Pi} = 10 mM
1-10.3. Summary of Tenets of the Chemiosmotic Model for Energy Coupling in

Biological Membranes. (Mitchell, 1966)
1. Energy transducing membranes are sealed and impermeable to coupling ions except for the
pathways involved in redox- and protein dependent ion translocation. The ions that have been
documented to couple energy transduction are H+ in most studies, and also Na+.
2. The high energy intermediate in membranes that can be used to sustain active transport or ATP
synthesis is the ion electrochemical potential, which for protons, is written as ∆µ̃ H + . ∆µ̃ H + is
determined by the trans-membrane pH gradient and membrane potential, which are energetically
equivalent forms of energy storage. The dependence is written quantitatively as
∆µ˜ H + = F ⋅ ∆ψ − 2.3 RT ⋅ ∆pH with the actual sign of the ∆Ψand ∆pH dependent on the initial
and final states for the ion movement.
3. The ATP synthase/ATPase activity, along with the transmembrane H+ flux, is reversible:
ADP + Pi + nH p + → ATP + H 2 O + nH n + , (i) with H+ flux from the p-side to the n-side of the
membrane coupled to ATP synthesis, and (ii) H+ flux from the n- to the p-side coupled to ATP
hydrolysis.
4. Non-protein uncouplers of membrane energy transduction that de-energize the membrane are
lipophilic (membrane-soluble) weak acids or bases that catalyze the equilibration of H+ or OH-
across the membrane.
Homework problem 17. How many protons must be translocated across (a) a small spherical
liposome (diam. = 300 Å)-neglect membrane thickness, and (b) a spherical E. coli cell of 1 µm
diam., in order to generate a trans-membrane potential of 100 mV? Assume both membranes have
the same specific membrane capacitance, 1µF/cm2.

1-10.4 Experimental Tests of the Chemiosmotic Hypothesis;
1-10.4.1 Proton Movement

+ +
H or Na movement driven by electron transport or ATP hydrolysis across a closed and
non-leaky organelle or bacterial membrane is always associated with formation of a ∆µ~ H+ across
that membrane. Proton efflux (as in Fig. 1-8B) driven by an O2 pulse delivered to anaerobic E. coli
cells is shown (Fig. 1-10).
(a) (b) (c)
0.001 pH 30 sec.
2.5 nequiv. H+
H+/O = 2.31
5 nequiv. H+
H +/O = 0.65
H +/O < 0.20

t1/2 = 11sec
+
Figure 1-10. H efflux induced by an O2 pulse (upward arrows) of air-saturated buffer in E. coli
+
cells, (a) untreated, (b) in the presence of uncoupler to short-circuit the H flow, and (c) in the
–
presence of thiocyanate (SCN ) as a lipid-soluble counter anion to prevent polarization of the
membrane (Gould and Cramer, 1977). A common pH scale, determined by adding known
equivalents of HCl to the cell suspension, is shown for (a, b). The slow irreversible responses in (a,
b) are slow"artificial background" responses that are not associated with redox-linked H+ pumping.
~ +
1-10.4.2 ATP Synthesis by an Artificial ∆µ H
A. The energetic ability of a trans-membrane ∆pH to support ATP synthesis was demonstrated by
incubation of chloroplast thylakoid membranes in the dark at pH 4 in the presence of a permeant
acid, followed by transfer to a solution containing ADP and Pi at pH 7. The membranes catalyzed
the synthesis of 140 nmol ATP/mg chlorophyll ATP in the dark acid-base transition compared to 5
– 10 nmol ATP/mg Chl in controls without ADP, phosphate, or (Jagendorf and Uribe, 1966).
Synthesis of ca. 150 nmol ATP/mg Chl corresponds to ~ 1 ATP per 6-7 chlorophyll molecules,
approximately 100 times the content of the chloroplast electron transport chain components
(stoichiometry approximately 1:600 Chl). Neither the presence of electron transport inhibitors, nor
the redox state of the chloroplasts, affected the amount of ATP synthesized during the dark acid-
base transition.

B. Artificial ATP Synthesis in Submitochondrial Particles (SMP)

SMP subjected to a ∆µ~H+ generated by an acid-base transition and a potassium diffusion
potential in the presence of ADP and 32Pi, can generate 2-3 nmol ATP/mg protein compared to 10%
of this amount in the control. The ∆µ~H+ including the diffusion potential is generated by diluting
+
SMP at pH 5.0 without potassium into a medium at pH 7.5 containing 100 mM K in the presence
+
of the lipophilic K –specific ionophore, valinomycin, MW = 1,110.0; cyclic peptide made of
repeating units of (L-lactate)-(L-valine)-(D-hydroxyvalerate)-(D-valine) (Fig. 1-11A). The
facilitated inward flow of K+ provides a ∆ψ to drive the H efflux (Thayer and Hinkle, 1975).
+
C. Reconstitution of Bacteriorhodopsin in Membrane Vesicles that Catalyze H+

Uptake and ATP Synthesis
Reconstitution of the purified bacteriorhodopsin pigment-protein from halophilic bacteria
+
into artificial membrane vesicles catalyzes H uptake (Racker and Stoeckenius, 1974).
Simultaneous incorporation of the mitochondrial ATP synthase into the vesicles (20 nmol ATP:mol
bR) catalyzed light-dependent ATP synthesis (15 mol ATP: mol bR), compared to 0.5 mol
ATP:mol bR in the control. The light-dependent ATP synthesis was sensitive to uncouplers (see
below) of membrane energy transduction.

(A) (B)
K+
hydration
high K+ shell
K+
H
CF3 O- -N-N=C(CN)2
low K+ K+
(C)
kA
A- A-
H+ H+
kHA
HA HA
~ ~
[µH+]n membrane [µH+]p
Fig. 1-11. (A) Schematic view of the ionophore, valinomycin (MW = 1,110, three repeating units
of [(L-lactate)-(L-valine)-(D-hydroxy isovalerate)-(D-valine)], as a mobile carrier for a dehydrated
monovalent cation. The valinomycin dodecamer can be thought of as a "doughnut," hydrophobic
outside with a central polar niche into which the cation can fit. The ion selectivity is Rb+ > K+ > Cs+
>Ag+ > NH 4+ > Li+. (B) Structure of the protonated form of the weak acid lipophilic uncoupler,
+
FCCP; (C) model for uncoupling action of weak acid anionic uncouplers that catalyze H flow in
the membrane from the side of positive µ~H+ to negative ~µ H+ [modified from (Benz and McLaughlin,
1983)].
D. Mechanism of Action of Uncouplers of Phosphorylation

It was proposed in the chemiosmotic hypothesis (Section 1-10.3) that weak acid lipid-
soluble uncouplers of ATP synthesis (e.g., carbonyl cyanide p-trifluoromethoxy-phenylhydrazone,
FCCP; pKa, 6.2 in 10% ethanol; Fig. 1-11A) act by increasing the conductance or permeability of
the membrane to protons.
Uncouplers increase the proton conductance of planar bilayer membranes by 2 – 3 orders
of magnitude, with the maximum increase occurring near the uncoupler pKa suggesting that the
unprotonated form of the uncoupler (e.g., FCCP) is translocated across the membrane in a shuttle-
like mechanism and thereby dissipates the ∆µ~ H+ (Fig. 1-11B). Uncouplers stimulate the rate of
respiratory and photosynthetic electron transport that is coupled to ATP synthesis by a factor of 2 –
10, depending on ambient pH and the tightness of coupling of the membranes. This phenomenon
is called “respiratory control” in mitochondria. The absence of mitochondrial respiratory control
and the presence of high respiratory electron transport rates are a frequent manifestation of human
mitochondrial myopathies (Luft et al., 1962).
Homework problem 18. ∆pH = 0 at pH = 7.5 in isolated right side-out E. coli membrane
vesicles. At this pH, the source of all of the free energy available for active transport and ATP
synthesis from a positive electrochemical potential gradient ( ∆µ̃ H+ = + 4 kcal/mol) generated by
respiration resides in the ∆Ψ. However, the ∆pH can be solely responsible (∆Ψ = 0) for transport
of some solutes at lower pH, e. g., pH 5.5. This is one indication that the ∆pH and ∆Ψ are
thermodynamically and enzymatically interchangeable. Calculate the value of the ∆ Ψ and the ∆pH
that can drive active processes at pH 7.5 and 5.5, respectively. T = 25 °C.
D. Mechanism of Action of Uncouplers (continued)
Fig. 1-12. Effect of ADP and uncouplers on rates of (A) respiratory electron transport and oxygen
consumption in mitochondria, and (B) photosynthetic electron transport and oxygen evolution in
chloroplasts.
1-11. ATP and “High-Energy” Bonds
The definition of a “high-energy” bond in biochemistry is that it is unstable and readily

hydrolyzed with a ∆G o at pH 7 ( ≡ ∆G o' ) more negative than that of the majority of simple
phosphate esters, i.e., ∆G o' ≤ –7 kcal/mol (Table 1-2). Most high-energy compounds involve
phosphate or sulfur anhydrides. High-energy compounds other than the phosphate anhydride
compounds shown in Table 1-2 include thiol acyl esters, sulfonium compounds, acyl imidazole, and
acyl amino acids. ATP occupies a central position in the hierarchy of high energy phosphoryl
compounds. Thus, the ADP/ATP couple can mediate phosphate flow from phosphorylated
compounds with very negative ∆G o′ (i.e., phosphoenolpyruvate, 1,3-diphosphoglycerate, creatine
phosphate) and can act as a phosphoryl donor or acceptor.
CTP, GTP, and UTP, which have free energies of hydrolysis very similar to that of ATP, are
preferentially used as the energy source for different biosynthetic pathways. They are the general
precursors, respectively, for biosynthesis of lipid, protein and cellulose, and polysaccharides.
The origins of the high-energy nature of ATP are: (i) the negative ∆H o from electrostatic
repulsion caused by the negative charges on the terminal β−γ phosphate groups, and (ii) the positive

∆S o arising from the increased number of substates or arrangements available in the phosphate
product due to resonance, and a decrease in the amount of H 2O solvated (and ordered) in the
products. The net charge is affected by the pK for HATP3– → H+ + ATP4–, which is approximately
6.8.
Table 1-2. ∆G o′ for hydrolysis of phosphate anhydride and ester compounds

Compound ∆G o (kcal/mol)a
Phosphoenolpyruvate –14.8
1,3-Diphosphoglyceric acid –11.8
Creatine phosphate –10.0
Acetyl phosphate –10.0
Phosphoarginine (pH 8.0) –8.0
ATP → ADP + Pi(+Mg ) ++
–7.7
ATP → ADP (pH 8.0) –8.4
ATP → ADP (pH 9.5) –10.4
Glucose-1-phosphate –5.0
Pyrophosphate –4.0
Fructose-6-phosphate –3.8
Glucose-6-phosphate –3.3
Glycerol-1-phosphate –2.2
a
pH = 7.0, except where noted.
The contribution of entropy or resonance stabilization to the large ∆G o of hydrolysis is a

consequence of the atomic properties of phosphorous, i.e., large bond lengths with weaker binding
energies. This leads to a partial double bond character of the orthophosphate P–O bonds. The
average P–O bond length in orthophosphate is 1.54 Å compared to 1.73 Å expected for a single P-
O bond. The greater electron delocalization is associated with resonance stabilization and a positive
contribution to the ∆S o of hydrolysis (Table 1-3). Another major contribution to the large positive
∆S o and negative ∆G o of hydrolysis of phosphate anhydride compounds arises from differential
solvation by H2O of products and reactants. The solvation energies of H2PO4–, HPO42–, and PO 43–
are 76, 299, and 637 kcal/mole (25oC). This implies that the large ∆S o of ATP hydrolysis arises
from a smaller amount of solvated or ordered water in the products because they carry a smaller
average charge than the reactants.

Table 1-3. Factors contributing to large negative ∆G o of hydrolysis of phosphate anhydrides such
as adenosine 5’-triphosphate (ATP)
Factor –∆H o or +T ∆S o
1. Electrostatic charge repulsion –∆ H o
2. Large number of resonant forms of orthophosphate + T ∆S o
3. Smaller amount of solvated or ordered water in less highly + T ∆S o
charged products
1-11.1 Experimental determination of the ∆G o for ATP Hydrolysis
At pH 8, in the presence of 10 –2 M Mg2+, ∆G o = –9 kcal/mol, and Keq > 106, which is too
large to be measured accurately through only the ATP hydrolysis reaction, ATP + H 2O → ADP +
Pi (See problem 19). Therefore, the Keq for ATP hydrolysis must be measured in two or more
coupled reactions such as the glutamine synthetase and glutamine hydrolysis reactions [Eqns (i)
and (ii)] below (Rosing and Slater, 1972). Thus,
K
(i) Glutamic acid + NH4+ + ATP  I → Glutamine + ADP + PI; KI = 700
K
(ii) Glutamine + H2O  
II → Glutamic acid + NH +; K = 230
4 II
The sum of reactions (i) and (ii) is:
ATP + H2O → ADP + Pi; with KΣ = KlKll; KΣ = 1.6 x 105 . Then,
∆G Σo = ∆G ATPo = ∆Glo + ∆Gllo = - RT 1nKΣ = 2.3RT⋅log KΣ = -30.5 kJ/mol.
Homework problem 19. (a) If 1 mM ATP is added to a solution at pH 8 in which it can be

hydrolyzed to equilibrium, calculate the concentration of ATP at equilibrium if it is assumed that
Keq = 106. (b) Briefly describe a method that could be used to determine the Keq for ATP
hydrolysis more accurately.
1-12. Oxidation-Reduction Reactions
1-12.1. Direction of redox reactions

As discussed in section 1-8.1, a free energy change, ∆G, results from movement of electrical
charge, nzF, through an electrical potential gradient. Thus,
∆G = + nzF ∆E = –nF ∆E, (1-62)

for the free energy change in a reversible reaction associated with electrical work driven by a change
in oxidation-reduction potential, ∆E , with z = –1 for electrons, and
∆G o = –nF ∆E o (1-62a)
for the changes in standard free energy and standard potential. Then, ∆Gfinal - ∆Ginitial =
∆G o = –RT 1nKeq, and (1-63)

RT 2.3 RT
EFo − EIo = ∆E o = • 1nKeq = log10 Keq = 59 log10 Keq (1-64)
nF F
(i.e., ∆E˚ = 59 log Keq)in mV when n = 1; 2.3RT/F = 59 mV at 25˚C.
Using the criteria that reactions at their midpoint go forward if ∆Go < 0 and away from the
midpoint when ∆G < 0 with ∆G = –nF∆E, the criterion for the forward or spontaneous direction of
an oxidation-reduction reaction is, respectively,
∆E > 0, or ∆Eo > 0 (1-65)
The criteria Keq > 1, ∆Eo > 0, ∆E > 0 are used to predict the direction of a redox reaction.
1-12.2 Properties of Oxidation-Reduction Potentials
A. Electron transfer tends to proceed in the direction of more positive E and E o.
B. A strong reductant results from a redox couple with a negative oxidation-reduction potential,
and a relatively strong oxidant from a redox couple with a positive potential.
C. Absolute values of the oxidation-reduction potential, like the absolute value of the free
energy, have no meaning in nature. The absolute values are set by the reference or
standard that is chosen, which in this case is the potential of hydrogen half-cell reaction:
2 H + + 2e − → H2 ( g) ; Eo = 0.0 V; pH = 0 (1 M H+).
D. Oxidation potentials, E and E o, are not state functions. A consequence is that these
functions are not necessarily additive. That is, for a series of sequential redox reactions
A→Β→C→D,
∆EAD ≠ ∆EAB + ∆EBC + ∆ECD, (1-66a)
although the free energy, as a state function, is additive,
∆GAD = ∆GAB + GBC + ∆GCD. (1-66b)
E. The oxidation-reduction reaction of a single redox-compound is written as a “half-cell”

reaction:
[A(ox) + e– → A(red); or for a pH-dependent reaction, (1-67a)
A(ox) + 2 e– + 2H+ → AH2] (1-67b)

The free energy changes associated with half-cell reactions are:
∆G o = –nFE o, (1-68a)
and
∆G = –nFE (1-68b)
1-12.3. The hydrogen half-cell. One can see from the hydrogen half-cell reaction,
2H+ + 2e- ↔ HL(g); E o = 0.0V, that its equilibrium constant and E o are both pH dependent. The
Eo’ standard potential (in mV) at pH = 7, E o’ = E o – 7 ( 5 9 . 1 ) = i s – 4 1 4 m V a t 2 5 o C ( c f . ,
p H d e p e n d e n c e o f m i d p o i n t p o t e n t i a l , s e c t i o n 1 - 1 2 . 9 b e l o w) . Using the hydrogen
electrode as the standard, a short list of half-cell redox reactions of interest to membrane energy
transduction is shown in Table 1-4.

Table 1-4. Oxidation-reduction potentials of some redox couples or half-cell reactions

important in bioenergetics
Midpoint potentials (mV)
Half-reaction E o (pH = 0) E o' (Em7)
Ferredoxin (ox) + e– → Fd (red) [2Fe–2s] ___ –432
2H+ + 2e– → H2(g) [Reference] 0.0 –414
NAD+ + H+ + 2e– → NADH –113 –324
FMN(ox) + 2e– + 2H+ → FMNH2 + 209 –205
.
O 2(1) + e– → O2– ___ –160
.
UQ + e– → UQ–
.
___ –150
cytochrome b + e– → cytochrome b (red) ___ –150 - + 50
ubiquinone + 2e– + 2H+ → ubiquinol ___ + 60
plastoquinone + 2e- + 2H+→ plastoquinol ___ + 90
cytochrome c1 +e– → cytochrome c1,(red) ___ + 220
cytochrome c + e– → cytochrome c(red) ___ + 250
cytochrome f + e– → cytochrome f(red) ___ + 370
plastocyanin + e– → plastocyanin(red) ___ + 370
Rieske [2Fe-2S](ox)+ e- → Rieske [2Fe- _ _ _ + 390

2S](red)
cytochrome a + e– → cytochrome a(red) ___ + 250–+ 400
Photosystem I-Chl (ox) +e- → Chl(red) ___ + 5001
O 2 + 4H+ + 4e– → 2H2O + 1 ,2 3 0 + 815
Photosystem II-Chl (ox) +e- → Chl(red) ___ + 1 ,2 0 0 2
1
Photosystem I, 2photosystem II reaction center Chl in oxygenic photosynthesis.
1-12.4 Oxidation-Reduction Potential as a Group-Transfer Potential;

Comparison of Standard Potentials and pK Values
The standard potential is a measure of the tendency to donate or accept electrons. The more
negative the potential, the greater the tendency of the reduced compound in a half-cell reaction (1-
65a) to donate (reducing ability), and the more positive, the greater the affinity for the electron of the
oxidized compound (oxidizing ability). Thus in Table 1-4, reduced ferredoxin and NAD(P)H are
strong reductants, and molecular O2 is a strong oxidant. Strong reductants and oxidants are good

electron donors and acceptors, respectively, just as strong and weak acids are good proton donors
and acceptors. The standard potential, E o, of an oxidation-reduction reaction is analogous to the pK
of an acid-base reaction (Table 1-5). As the pK is the pH at which an acid is half-protonated, the E o
is the redox potential under standard conditions at which an electron donor-acceptor is 50%
reduced. For this reason, the notation Em is often used instead of Eo to designate the midpoint
potential. The Em becomes Eo under standard conditions.
Table 1-5. The oxidation-reduction potential as a group transfer potential.
Proton-transfer potential Electron-transfer potential

(acid-base) (redox potential)
Reaction A- + H+ → HA A+ + e- → A
Measure of transfer ∆G 0 ∆G 0
potential pKa = E0 = −
2.3 RT nF
1-12.5 Dependence of ∆E on Reaction Pathway: Reduction of O2 to H2O

∆E , unlike ∆G, is not a state function and depends on the path of the reaction. As an
example, consider the four different pathways for reduction of O 2 to H 2O involving the
.
intermediates, superoxide (O2–) and hydrogen peroxide (H2O2), A→B→C→D (Fig 1-12). One
path would be the four-electron reduction:
O2 + 4e- + 4H+ → 2H2O, with the midpoint potential at pH 7, Em7 = 0.82V.

The relationships between the Em7 values for the different reactions can be determined from Eqns
.
(1-66a) and (1-68a). The value for one of them [e.g. EAB = Em7(O2/O2–)] can be solved from the Em7
.
and n values for the O2/H2O (EAD = 0.82 V, n = 4) and O2–/H2O (EBD = 1.20 V, n = 3) couples. EAB
. .
(O2 → O2–) can be obtained from EAD (O2 → H 2O) and EBD (O2– → H 2O) because ∆GAD = ∆GAB
+ ∆GBD. One can write the half cell reactions as:
O 2 + e − → O 2−˙ E AB = ? (1e - reaction)

O 2−˙ + 3e − + 4H + → 2H 2 O E BD = +1.20 V (3e - reaction)
O 2 + 4e − + 4H + → 2H 2 O E AD = +0.82 V (4e - reaction)
From
∆G oAD = ∆G oAB + ∆G oBD , substituting for ∆G
,
−4F ⋅ E oAD = −(1)F ⋅ E oAB − (3)F ⋅ E oBD ; solving for E oAB
(1 + 3)(0.82 ) − (3)(1.2 )
E o AB = = −0.32 V
1

+0.31
-0.3 3 . +0.94 +1.33

O2 O2 - H2 O2 H2 O
+1.20
+0.82
Figure 1-12. Standard reduction-oxidation potentials for the intermediates involved in the reduction
of O2 to H2O at 25 C and pH 7.0. The pathway, O 2 → O 2−̇ → H 2 O 2 → H 2 O is designated A → B
→ C → D in the text.
1-12.6 Reduction of compound B by A
A(ox) + n1e- → A(red), Eo = EAo; G o = GA o (1-69a)
B(ox) + n2e- → B(red), Eo = EBo; G o = GB o (1-69b)
with n2 ≠ n1 the charge balanced reaction is,
 n2   n2 
  A(red ) + B(ox ) →   A(ox ) + B(red ) ,
 n1   n1 
for transfer of n2 electrons from A to B. The standard free energy change, ∆GABo, for transfer of the
n2 electrons from A to compound B that requires 2 electrons for reduction is:
n 
∆GABo
= GBo −  2  GAo . (1-70)
 n1 
Using Eqn 1-60, and substituting into eqn. (1-70),
− n2 F ⋅ ∆EAB
o
( n
)
= − n2 F ⋅ EBo − 2 − n1F ⋅ EAo ,
n1
it follows that:
∆EABo
= EBo − EAo , (1-71a)
which is the standard potential of electron acceptor minus that of the donor. Similarly,
∆EAB = EB − EA , (1-71b)
so that the change of standard potential in a complete redox reaction is the difference of the
standard potentials for the two half-cell reactions, as discussed in section 1-12.1. For the reaction
to proceed in the forward direction, ∆E > 0 and EBo > EAo, i.e., in the direction of more positive Eo
and higher electron affinity.
1-12.7 Concentration dependence of the oxidation-reduction potential

 {c} 
From G = G o + RT ln o  ,
 {c } 

Π{ products}
∆G = ∆G o + RT ln , where the brackets indicate activities; then,
Π{reac tan ts}
because ∆G = − nF ⋅ ∆E , and
∆G o = − nF ⋅ ∆E o
for the reaction, [ox ] + ne − → [red ] , involving the transfer of n electrons, it follows:
∆G o RT Π{ prod}
∆E = − − ln
nF nF Π{react}
RT Π{ prod}
= ∆E o − ln
nF Π{react}
RT Π{ prod}
= ∆E o − 2.3 log10 . (1-72)
nF Π{react}
For a half-cell reaction, one would write:
RT Π{ prod}
E = E o − 2.3 log10 (1-73)
nF Π{react}
From (1-72), since 2.3RT/F = 59.1 mV at 25 C,

59 Π{ prod}
∆E ( mV ) = ∆E o − log10 (1-74)
n Π{react}
and for a half-cell reaction,

59 {red}
E = Eo − log10 (1-75)
n {ox}
The fact that the Eo values can be considered midpoint potentials when there is no pH or
ligand dependence of the Em, and at dilute concentrations of reactants and products, is illustrated in
Eqns. (1-73) and (1-74). It can be seen that E = Eo and ∆E = ∆Eo when the activities of the products
or reduced species equals those of the reactant or oxidized component of the half-cell. Thus,
59 Π{ prod}
∆E = ∆E m - log10 (1-74a)
n Π{react}
59 {red}
E = Em - log10 (1-75a)
n {ox}
As discussed above (Section 1-7.4) for ∆G, the direction of an oxidation-reduction reaction is
determined by the concentrations of products and reactants as well as by the ∆Em, i.e., by the ∆E.
Note: In the context of the group transfer potential (section 1-12.4), compare (1-75a) to the
Henderson-Hasselbach equation for H buffering: pH = pK + log

+
A−
.
{ }
{ AH}

1-12.7.1 Concentration dependence in donor-acceptor reactions
For the reaction A(red) + B(ox) →A(ox) + B(red); 1 e- reaction (n = 1),
A(ox ) B(red )
∆E = ∆Em − 59 log ⋅ , (1-75b)
A(red ) B(ox )
for every 10-fold increase in the ratio of A(red)/A(ox), or B(ox)/B(red), the ∆E will increase by +59
mV, and the ∆G for the reaction will be more negative by –5.69 kJ/mole.
1-12.8 Graph of titrations of the oxidation-reduction reaction
For a half-cell reaction involving transfer of one e- (n = 1), e.g. cytochrome c,

cyt c(ox ) + e − → cyt c(red )
E = Em − 59 log
{cr } .
{co }
Note that for every 10-fold increase in c(red)/c(ox), E decreases by 59 mV. (Note the analogy with
acid-base problems). Then,
1
E = Em − 59 log −1 ; {cr } + {co } = {cT } , the total concentration (1-76)
 cr 
  −1
 cT 
for n = 1 at 25 C, in mV; cT = total cytochrome concentration (reduced + oxidized), cr/cT = fraction
cytochrome reduced.
Two aids in graphing the titration are: (i) cr = co and cr/cT = 0.5 when E = Em, (ii) cr/co
changes by a factor of 10 for every change of 59 mV in the potential. Thus, cr/cT = 0.5 and co/cr = 1,
when E = Em. cr/cT = 0.91 and cr/co = 10 when E = Em – 59 mV; cr/cT = 0.09 and cr/co = 1/10 when E
= Em + 59 mV (Fig 1-13A). The actual titration involves measuring changes in absorbance at a
convenient wavelength (e.g. 550 nm) of the cytochrome as it is progressively reduced and oxidized.
The spectrum of the reduced protein, with sharp peaks at 417 and 550 nm, and that for the oxidized
cytochrome, relatively flat in the region of 550 nm, of horse heart cytochrome c, are shown in Fig 1-
13B.
For a redox titration of compound “X” using visible spectroscopy, the titrations are
commonly made in a stirred cuvette specially fitted with (a) a redox combination electrode (Pt-
Ag/AgCl), (b) a port to allow addition of a strong oxidant and strong reductant to change the
potential (analogous to addition of acid and base in a pH titration, and (c) an entry and exit port for
N 2 or Ar gas for anaerobic titrations of reducing systems with a negative (reducing) potential that
can be readily oxidized by O2. The solution in the cuvette with compound “X” will contain redox
buffers (Table 1-6) whose Em7 values cover the range of E values over which the titration wil be
measured. Redox buffers are analogous to the need for pH buffers when performing pH titrations.
As a pH buffer that undergoes a 1 H+ protonation-deprotonation will buffer over a range of ±1 pH
unit around its pK, a redox buffer undergoing a 1 e- oxidation-reduction will buffer over a range of
±59 mV around its Em.

cr = co
1.25
1 n = 2
0.75 n = 1
E - Em = 0
c r/ cT
0.5
0.25
-0.25
-200 -150 -100 -50 0 50 100 150 200
(E - Em )
(A)
(B)
Figure 1-13. (A) Determination of the midpoint potential of cytochrome c by titration of the
ambient potential. The Fe3+ -heme ⇔ Fe2+ - heme titration of cyt c involves the transfer of one
electron (n = 1) in the Nernst equation (eqns 1-72 to 76). An n = 2 titration, e. g., for the H 2
electrode or quinone ⇔ quinol is also shown. (B) Reduced (peaks at 417 and 550 nm) and
oxidized spectra of horse heart cytochrome c.

Table 1-6. Some redox buffers and their midpoint potentials.

Redox buffers Em7 (mV)
Anthraquinone-2-sulfonate -220
2-OH-1,4 naphthoquinone -140
2,5-dihydroxy-1,4-benzoquinone -60
1,4-naphthoquinone +65
1,2-naphthoquinone +135
Homework problem 20. (A) Using values of R and F in appropriate units, evaluate the coefficient
2.3RT/nF (in millivolts) at 5°C and 25°C for n = 1 and n = 2.
(B) Consider the half-cell oxidation-reduction reaction, of redox compound, r: r(ox) + e– + H + →
r(red). If the midpoint potential, Em, for this reaction = 0.0 V at pH 7,
(a) What is the value of the potential, E, for the reaction at pH 7, when the quotient of concentrations
(activities) is:
r (red) r (red) r (red) r (red)
(i) = 10; (ii) = 1000 (iii) = 10 −1 ; (iv) = 10 −2 .
r (ox) r (ox) r (ox) r (ox)
(b) What is the Em for the half-cell reaction at pH 4?
Homework problem 21. The half-reactions of cytochromes b and c are (n = 1):
b(ox) + e- → b(red) Eo = -0. 035 V

c(ox) + e → c(red) Eo = +0.26 V.
(a) If these two redox centers are electronically connected, what is the equilibrium constant for
(a) the reduction of cytochrome c by cytochrome b ;(b) For reaction (a), what is the
potential change, ∆E, for the reaction when the ratio of reduced to oxidized cytochrome is:
b(red): b(ox) = c(ox): c(red) = 10:1, or (d) = 1:10? T = 25 °C.
Homework problem 22. How much energy, in eV and kJ, is stored at pH 7 in reducing
equivalents when two electrons are transferred from the special pair bacteriochlorophyll (Em = +470
mV) of the bacterial photosynthetic reaction center to the secondary quinone, QB, whose Em7 = + 50
mV?
12.9 Half-cell Potential for a Reaction Involving e– (n = 2) and H+ transfer;

e.g., Reduction of Quinone to Quinol
The half cell-reaction for the reduction of the aromatic quinone to quinol (Fig. 1-14A) is:
Quinone + 2e– + 2H+ → Quinol (QH2); n = 2 (1-77)
Using the standard potential at pH = 0 as a reference, (from 1-75, 1-75a)

59 {QH 2}
E = Eo – log , (1-78)
2 {Q}{H +}2
or
59 59 {QH 2}
E = Eo + log{H +}2 – log ;
2 2 {Q}
then,

59 {QH 2}
E = (E o – 59 pH) – log
2 {Q}
and
59 {QH 2}
E = Emh – log , where (1-79)
2 {Q}
(Emh = E o – 59 pH), and (1-80)
Emh is the midpoint potential at a particular pH value. Notes: (a) The standard potential, E o, of the
hydrogen electrode is defined as 0.0 V at pH 0, 1 atm pressure, and 20oC. (b) The slope of the n =
2, Q ↔ QH2, titration is steeper than that for n = 1. Thus, whereas the potentials (in mV), relative to
the Em, at which the n = 1 compound is ~90% oxidized or reduced are (Em + 59) and (Em – 59),
respectively, the corresponding potentials for the n = 2 compound are (Emh + 29.5) and (Emh –
29.5) [cf., Fig. 1-13A].
O OH
+ 2H + + 2e -
O OH
(A)
O OH
R2 R1 H3 C CH3
CH3
H3 C CH3
R3 (CH2 -CH=C-CH2 ) nH
O OH
(B) (C)
Fig. 1-14. (A) Reduction of benzoquinone to hydroquinone.

(B) Structure of the lipophilic ubiquinone (n = 6-10; R1 = CH3; R2, R3 = CH30),

found in mitochondrial and bacterial membranes, or plastoquinone (n = 9; R1 = H;
R2, R3 = CH3) found in chloroplast and other oxygenic photosynthetic membranes.
(C) The tetramethyl-substituted quinol (duroquinol) with an Em = +480 mV.
1-12.9.1 Total pH dependence of Em of a redox prosthetic group

The above expression (1-80) for reduction of a quinone to quinol is valid throughout the
physiological range. However, at extremes of low and high pH, the Em will not obey eqn (1-80) and
will become pH-independent (Fig. 1-15). This is because at the low and high pH extrema,
respectively, (a) the oxidized form of the redox compound is protonated, or (b) the reduced form in
deprotonated. Thus, at the low and high pH extrema, the Em reaches asymptotic constant values of
EmAcid and EmBase (Fig. 1-15). At intermediate pH values, the Emh depends on pH as in eqn (1-80).
The protonation-deprotonation of the oxidized and reduced forms at low and high pH, respectively,
is described by pK values, pKox and pKred.
500
Em,acid Em = Eo - 59•pH
450
400
350 p K ox = 3
Em h
300 p Kred = 7
250
200 Em,base
150
-2 0 2 4 6 8 10 12 14
pH
Fig. 1-15 pH dependence of Em of redox prosthetic group including effects of pK ox and pKred
(pKred>> pKox) [modified from (Dutton, 1978)].
The pH cycle of proton and electron transfer reactions can then be described by the scheme
in Fig. 1-16.

e-
Red - H Ox - H
H+
pKred pKox
H+ e-, H+
Red Ox
e-
Fig. 1-16. Cycle of pH-dependent electron transfer reactions. The top and bottom lines in the cycle
describe the redox reaction at the extreme of low and high pH, respectively. The pH-dependent
reaction in the diagonal operates at intermediate pH values. Some groups whose protonation-
deprotonation are linked to an oxidation-reduction are histidine ligands of hemes and iron-sulfur
proteins, and the heme propionate side chain. (modified from (Dutton, 1978).
The analytical expression for the complete pH dependence of the Em can be derived as
follows:
{ }
{ox} H +
= Kox ;
{red} H + { }
= Kred (1-81)
{oxH} {redH}
Eh = Emh − 59log
{totalred } (1-81a)
{totalox }
{red + redH}
= Emh − 59log (1-81b)
{ox + oxH}
since {totalred} = {red} + {red • H+}, and
+
{totalox} = {ox} + {ox • H }; then, from (1-81),
{redH}
{red} = Kred
{H } +
{oxH}
{ox} = Kox ; then, substituting into (1-81b),
{H } +
Kred
redH ⋅
+ redH
Eh = Emh – 59 log H+ (1-81c)
K
oxH ⋅ ox+ + oxH
H
Kred
redH (1 + H + )
then, factoring, Eh = Emh – 59 log (1-81d)
oxH (1 + Kox )
H+

K
1 + red
= Emh – 59 log
redH
– 59 log H+ (1-81e)
oxH K
1 + ox+
H
{redH}
For pH << pKox, pKred, [H+]>> Kox, Kred, Eh = Emacid − 59 log and
{oxH}
{redH}
−59 log = Eh − Emacid ; substituting into (1-81e)
{oxH}
Kred
1+
Eh = Eh + Emh – Emacid – 59 log H+ (1-81f)
K
1 + ox+
H
Then, rearranging terms in (1-81f):
K red
1+
E mh = E macid + 59log H+ ; (1-81g)
K
1 + ox+
H
Kox
1+
Emh = Emacid – 59 log H+ (1-82)
K
1 + red+
H
Formula (1-82) describes the complete pH dependence of the Em (Dutton, 1978). It can be
seen that Emh has the following values or pH dependence for the three regions of pH (a–c below)
(a) pH << pKox << pKred: Emh=Emacid (1-82a)
Kox
(b) pH >> pKred >> pKox: Emh = Emacid – 59 log (1-82b)
Kred
Kox
(c) pKox << pH <<pKred: Emh = Eacid − 59 log = Emacid − 59 pH − 59 log Kox (1-82c)
{ }H+
Homework problem 23. If the pKox and pKred of cytochrome x are 3 and 10, respectively, and
Emacid = +300 mV, what is the Em7 of the cytochrome?
Homework problem 24. (A) If Em7 = +65 mV for the mitochondrial n = 2 ubiquinone/ubiquinol
couple, what is the value of E at pH = 7 at which it is (a) 50% reduced, (b) 90% reduced, (c) 9%
o
reduced? (B) If the Em7 = +260 mV of benzo-hydroquinone (fig. 1-14A), what is its E ?

Homework problem 25. (a) Calculate the Em7 of the two semiquinone redox pairs that interact on
the p-side quinone binding niche of the mitochondrial membrane if the semiquinone formation
constant = 10-2 and the Em7 of the UQ/UQH2 couple is =65 mV. (b) What is the semiquinone
formation constant that will allow formation of a semiquinone whose Em1 at pH 7 equals that of one
of the hemes with Em7 = -112 mV of the integral membrane b cytochrome in which the quinone
niiche is embedded? [Thus, the semiquinone can readily reduce the b heme, which is a
demonstration of “oxidant-induced reduction” ].
Homework problem 26. If the redox span of the mitochondrial respiratory chain extends from
–320 mV [Em7 (NAD+/NADH)] to + 815 mV [Em7 (O2/H2O)], (a) calculate the ∆G made available
from the transfer of 2 electrons from NADH to O 2 with the electron donor and acceptor operating
at their mid-point potentials. (b) Calculate the number of ATP molecules that can be synthesized
per pair of electrons transferred down the respiratory chain from NADH to O 2 if ∆Go ATP = 33.5
kJ/mol, the ATP/ADP ratio = 100, and the phosphate concentration is 10-2 M.
1-12.10 Redox Properties of Semiquinones

In addition to the two-electron oxidation-reduction of quinines (1-77), one-electron
reactions involving the half-reduced semiquinone form of the quinone-quinol couple also occur
(Rich and Bendall, 1980).
If the two electron quinone reaction is:
Q + 2e – + 2 H + ↔ QH2 for the neutral quinol, and (1-83a)

Q + 2e – + H + ↔ QH − , for the anionic quinol (1-83b)
with midpoint potential ≡ Emh, then the reactions involving the semiquinone are:
Q + e – + H + ↔ QH • ; neutral semiquinone (1-83c)
Q + e – ↔ Q −̇ ; anionic semiquinone (1-83d)
with midpoint potential, Em1, and

QH • + e – + H + ↔ QH2 , (1-83e)
Q −̇ + e – + H + ↔ QH − (1-83f)
with midpoint potential ≡ Em2.
The other reaction linking the fully reduced (quinol), oxidized (quinone), and half-reduced
(semiquinone) components is that of semiquinone “dismutation” into the oxidized quinone, Q,
and the reduced quinol, QH2:
Q + QH2 ↔ 2 QH • , (1-83g)
−
Q + QH ↔ 2Q + H −
˙ +
(1-83h)
The equlibrium constant, Ks, for semiquinone formation,

Ks = 
( )
 QH • 2 

 ,or (1-83i)
 (Q)(QH2 ) eq

Ks = 
 ( )( )
 Q −˙ 2 H + 

− 
(1-83j)
 (Q) QH ( eq
)
is a measure of the stability of the semiquinone.
. .
The pK values of (QH2 → QH – + H+) and (QH → Q_ + H+) are approximately 11 and
5–6 respectively, for duroquinol and durosemiquinone (Fig. 1-14c).
The midpoint potential of the two electron quinone reduction is, in general, not equal to
those of the individual one electron reactions, i. e., ,Emh ≠ Em1 ≠ Em2, for the different quinone redox
reactions because the affinity of the quinone (Q) for one electron reduction to the semiquinone
.
(QH ) is different from its affinity for cooperative reduction to the quinol (QH2) by two electrons,
and also different from the affinity of the semiquinone for one electron reduction to the quinol. If
. .
the Em of the (Q/QH ) reaction decreases, then that of the (QH /QH2) reaction must increase to
balance the free energy change for the net (Q/QH2) reaction. This follows from the fact that the
pathway from the quinone through the semiquinone to the quinol is a second route for quinone
reduction to the quinol through two one-electron reactions, from a common initial state (quinone) to
.
the same final state (quinol). In addition, the more unstable the QH (smaller value of KS), the
. . .
stronger the reducing nature of QH or Q_, and therefore the more negative the Em of Q/QH .
Thus, there should be some definite relations between Emh, Em1, Em2, and KS.
To derive the relationships between Em1, Em2, and K2, let E1 and E2 be the working potentials
of the one electron reactions reactions (1-83d) and (1-83f). Then,
E1 = Em1 − 59 log
Q −˙ { }
{Q}
E2 = Em 2
{QH } .
− 59 log −
−
{Q ˙ }{H } +
Using a condition at which a relationship can be found between the constants, at equilibrium, E1 =
E2,
and, Em1 − 59 log

{Q −˙ }
= Em 2 − 59 log
{QH } −
; rearranging terms,
{Q}
eq
{Q−˙ }{H } +
eq
{Q −˙ } {H }
2 +
Em1 - Em 2 = 59 log = 59 log Ks , (1-84)

{Q}{QH } −
eq
Where KS is the semiquinone formation constant.

If KS > 1, indicating that the semiquinone species is stable, Em1 > Em2, and the couple
( )
Q / Q −̇ operates at a more oxidizing (more positive) potential than the couple QH /QH2.
.
However, if K <1, and the semiquinone is unstable, then E < E , and the Q / Q −̇ couple
S m1 m2 ( )
.
provides a stronger reductant than the QH /QH2 couple. In the case of semiquinone species
thought to interact with proteins, the KS has been found to be 10–1–10–2 in mitochondria and

chromatophores, which corresponds to Em1–Em2 = –60 to –120 mV and a significant concentration

of semiquinone.
1-12.10.1 Relationship of Midpoint Potentials of the Quinone/Quinol n = 2

Reaction (Emh) and the Semiquinone n = 1 Reactions (Em1, Em2)
To determine the relationship of the midpoint potentials of the reactions described in Eqns. (1-83a-
f) , note that free energy changes, ∆G1 and ∆G2 are associated wih the one electron reactions,
Q + e – ↔ Q −̇ (1-83d)
−̇
Q + e + H ↔ QH ,
– + −
(1-83f)
Because the two electron reaction described in Eqn (1-83b) is the sum of (1-83d) and (1-83f),
Q + 2e – + H + ↔ QH − (1-83b)
∆G(Q/QH − ) = – nF ⋅ E(Q/QH − ) = – 2 F ⋅ E(Q/QH − )
= − F ⋅ E(Q / Q −˙ ) − F ⋅ E(Q −˙ / QH − )
= ∆G(Q/Q −˙ ) + ∆G(Q −˙ /QH − )

= − F  Em1 – 59 log10
{ } − F E
Q −˙
− 59 log
{QH } 
−
,
 {Q} 
{H }{Q } 
−
m2 .
   + ˙

and combining the arguments of both log terms,
-2 F ⋅ E(Q/QH ) = – F( Em1 + Em 2 ) +
-
59 F log
{QH }−
; dividing both sides
{Q}{H } +
by -2F,
E(Q / QH ) =− Em1 + Em 2 59
− log
QH −{ ;
} (1-85)
2 2 {Q} H + { }
The relation between the different midpoint potentials is then:
( Em1 + Em 2 )
Em (Q/QH2 ) = ; (1-86)
2
the midpoint potential of the two electron reaction is then the average of the two one-electron
semiquinone reactions.
Then, eqns. (1-84) and (1-86) are the two equations that allow determination of the two
unknowns, Em1 and Em2 in terms of Em, the midpoint potential of the 2-electron quinone reduction,
and Ks, the semiquinone formation constant. The two cases are:
(A) KS <1, then from Eq. 71, Em1 < Em < Em (1-87)

so that the strength of the reductant in the semiquinone/quinone couple > that of quinol/quinone >
that of quinol/semiquinone. Similarly,
(B) KS >1, Em1 > Em > Em2. (1-88)
From Eqns. (1-84) and [1-(86-88)], it can be seen that the splitting of Em1 and Em2 around
Em is symmetric, so that the two cases described in Eqns.[1-(88-89)] can be arranged in an Em level
diagram (Fig. 1-17). The more reducing potential of Em1 for the (Q / Q −˙ ) couple when Ks < 1 (Fig
1-16A) has biological importance because such a reducing semiquinone species is generated when
ubi- or plastoquinol is oxidized by the cytochrome bc1 or b6f complex in respiratory and
photosynthetic electron transport (Mitchell, 1976).
(A) (B)
(-)
E m1 E m2
E mh E mh
E m2 E m1
(+)
Figure 1-17. Relative Em values for KS < 1 (A) and KS > 1 (B); |KS| the same in (A), (B).
Homework Problem 27. Determine the midpoint potential of a mitochondrial cytochrome b heme
if it was found through titration of its optical spectrum at 25 °C to be 90 % and 99 % reduced at
potentials of –150 mV and – 209 mV, and 90% and 99 % oxidized at –32 mV and +27 mV.
Homework Problem 28. Calculate the Em7 of the two semiquinone redox pairs that interact at the
p-side plasto quinone binding site of the chloroplast membrane if the semiquinone dismutation
constant = 10-2 and the Em7. of the PQ/PQH2 redox couple is + 80 mV; temperature = 25°C.
_
Will the Em1 of the PQ/PQ be favorable for reduction of the p-side heme of cytochrome b6
whose Em7 = -50mV?
Homework Problem 29. The synthesis of ATP in mitochondrial membranes can be coupled to the
oxidation of the anionic quinol [Em (QH./QH– ) = + 209 mV] by the high potential iron-sulfur
protein, and subsequent electron transfer to a metal center of cytochrome oxidase, [Em = 491
mV]. If all of the free energy made available in the electron transfer from the quinol to the
oxidase is converted to a proton electrochemical potential, ∆µ̃ H +
(i) What is the value of the ∆G for consecutive transfer (n =1) of two electrons, with a ratio
of reduced: oxidized quinone = 10:1, and of reduced: oxidized oxidase = 1:10?
(ii) Calculate the efficiency for conversion of redox energy to ATP synthesis at 25o C, made
available from the transfer of two electrons, if the ATP/ADP poise is 100, the phosphate
concentration is 10–2 M, and ∆GoATP = + 8 kcal/mol.

1-13. Free Energy of Activation for Electron Transfer
Consider the transfer of an electron from a donor, D, to an acceptor, A:

D + A → ( D− −A) → ( D−A− ) → D + A− , where the docked donor (reactant) and acceptor
−
(product) complex marked in parentheses are included in the reaction. An activation energy for the
electron transfer arises because (a) the electron in the donor complex has a different potential and
free energy than the acceptor complex; (b) the transfer of the electron results in a change of the
internuclear distances in the atoms that surround the electron, and (c) a “reorganization energy”.
As shown in Fig 1-18, the reorganization energy, λ, is the energy that is required to change the
average internuclear distance, XDo, of the reactant, to that XAo, of the product complex without
electron transfer. This can be described in a diagram showing the energy of the electron in the
donor and acceptor complex, which moves in the electrostatic field created by the nuclei, as a
function of the average internuclear distance in the donor (D) and acceptor (A) complex (Figure 1-
18).
The GD and GA curves in Fig 1-18 are assumed to be equal parabolae corresponding to
oscillators with the same frequency. The equation of the two curves are (DeVault, 1984):
GD = kH
(X − X ) o 2
D
+ GDo (1-89a)
2
GA = k H
(X − X ) o 2
A
+ GAo , (1-89b)
2
with kH = force constant (Hooke’s law) of the parabolae. From Figure 1-18, measuring from XDo
(i) λ = kH
( ∆X )
o 2
, (1-90)
2
and (ii) ∆G = kH‡ ( ∆X )

‡ 2
; (1-91a)
2
(∆X )
2
o
− ∆X ‡
then, measuring from X ,A
o
(iii) ∆G = kH‡
+ ∆G o (1-91b)
2
with ∆G = GAo − GDo . Combining (i), (ii), and (iii) gives
(iv) ∆G ‡
=
(λ + ∆G ) o 2
. (1-92),
4λ
the activation energy, sometimes called the Franck-Condon term.

GD
GA
GD GA
Free Energy
∆G ‡
o
GD
∆X‡
∆G o
G Ao
∆Xo
XDo Xo XAo
Average Inter-Nuclear Distance
Figure 1-18. Free energy of the electron in the donor (D) and acceptor (A) complexes as a function
of the average internuclear distance. XDo, XAo are the inter-nuclear distances for the donor and
acceptor complexes at equilibrium, and ∆X≠, the distance change associated with the gain of
( )
activation energy, ∆G‡, by the donor complex. X Ao − X Do = ∆X o ; λ = reorganization energy.
(modified from (DeVault, 1984)).
Activation-less electron transfer. It can be seen from Eqn (1-92) that ∆G‡ = 0, and the electron
transfer is activation-less, when ∆Go = -λ. In principle, λ can be determined by measuring the rate
of electron transfer as a function of ∆Go that gives the maximum rate.
“Inverted region.” When -∆Go > λ, ∆G‡ ≠ 0, and the cross-over coordinate Xo, of the GA and GD
energy curves is at a position Xo < XDo.
Under conditions where the electronic coupling between donor and acceptor is relatively
weak, the rate constant for electron transfer, ket, is proportional to (a) the “Franck-Condon” term
that includes the effect of the activation energy, Eqn (1-93), and as well (b) to a proportionality
factor A(T) that is a function of temperature, and a function e-βd, that describes the dependence on
donor-acceptor distance, d. The distance decay constant, β, depends upon details of local structure.
Thus,
ket = A(T ) ⋅ exp( − βd ) ⋅ exp( − ∆G‡ / kBT ) (1-93)

where kB = Boltzmann’s constant, 1.38 x 10-23 J K-1. Further reading on the above aspects of
intraprotein electron transfer (Marcus and Sutin, 1985; Gray and Winkler, 1996; Page et al., 1999).
Homework Problem 30. Tabulate or graph the dependence of the relative rate of electron transfer
o
between electron donor and acceptor as a function of the ∆G for the transfer between them, varied
from +0.1 eV to –1.0 eV, with the reorganization energy, λ, = 0.6 eV. Assume all other parameters
of the electron transfer, including donor-acceptor distance, are constant. For what value of ∆Go is
the rate a maximum? Sketch approximate Marcus potential energy diagrams for reactant (R) and
o
product (P) states for ∆G = + 0.1 eV, – 0.6 eV and – 1.0 eV. (The main parameter of interest is
‡
∆G ).
Homework Problem 31. Calculate the activation energy for electron transfer (a) from the quinone,
o
QA, to quinone, QB, if the ∆G /F for the reaction = +0.1V; and from bacteriopheophytin to QA if the
o
∆G /F = – 0.7 V. Assume that the reorganization energy, λ, in both cases = +0.6 V.
References:
1. Benz, R., and S. McLaughlin. 1983. The molecular mechanism of action of the proton
ionophore FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone). Biophys. J. 41:381-
398.
2. Chandrasekhar, S. 1957. An Introduction to Stellar Structure. Chap. 1, Dover, New York.
3. Cramer, W. A., and D. B. Knaff. 1991. Energy Transduction in Biological Membranes.
Springer-Verlag, New York.
4. DeVault, D. 1984. Quantum-Mechanical Tunneling in Biological Systems. Cambridge
University Press, Cambridge.
5. Dill, K. A. 1997. Additivity principles in biochemistry. J. Biol. Chem. 272:701-704.
6. Dutton, P. L. 1978. Redox potentiometry: determination of midpoint potentials of oxidation-
reduction components of biological electron transport systems. Meth. Enz. 54:411-435.
7. Eisenberg, D. S., and D. M. Crothers. 1979. Physical Chemistry with Applications to the Life
Sciences. Benjamin Cummings, Menlo Park.
8. Gould, J. M., and W. A. Cramer. 1977. Relationship between oxygen-induced proton efflux
and membrane energization in cells of Escherichia coli. J. Biol. Chem. 252:5875-5882.
9. Griko, Y., S. D. Zakharov, and W. A. Cramer. 2000. Structural stability and domain
organization of colicin E1. J. Mol. Biol. 302:941-953.
10. Hill, T. L. 1966. Lectures on Matter and Equilibrium. W. A. Benjamin, New York.
11. Jagendorf, A., and E. Uribe. 1966. ATP formation caused by acid-base transition of spinach
chloroplasts. Proc. Natl. Acad. Sci. USA. 55:170-177.
12. Krishtalik, L. I., and W. A. Cramer. 1995. On the physical basis for the cis-positive rule
describing protein orientation in biological membranes. FEBS Lett. 369:140-143.
13. Luft, R., D. Ikkos, G. Palmieri, L. Ernster, and B. Alzelius. 1962. A study of severe
hypermetabolism of non-thyroid origin with a defect in the maintenance of mitochondrial
respiratory control: a correlated clinical, biochemical, and morphological study. J. Clin. Invest.
41:1776-1804.
14. Mahan, B. H. 1964. Elementary Chemical Thermodynamics. W. A. Benjamin, New York.
15. Mitchell, P. 1966. Chemiosmotic coupling in oxidative and photosynthetic phosphorylation.
Biol. Rev. Cam. Phil. Soc. 41:445-502.
16. Mitchell, P. 1976. Possible molecular mechanisms of the protonmotive function of cytochrome
systems. J. Theoret. Biol. 62:327-367.
17. Racker, E., and W. Stoeckenius. 1974. Reconstitution of purple membrane vesicles catalyzing
proton uptake and adenosine triphosphate formation. J. Biol. Chem. 249:662-663.

18. Ramos, S., and H. R. Kaback. 1977. The relationship between the electrochemical proton
gradient and active transport in Escherichia coli membrane vesicles. Biochemistry. 16:854-859.
19. Rich, P., and D. Bendall. 1980. The kinetics and thermodynamics of the reduction of
cytochrome c by substituted p-benzoquinols in solution. Biochim. Biophys. Acta. 592:506-518.
20. Rosing, J., and E. C. Slater. 1972. The value of ∆Go for the hydrolysis of ATP. Biochim.
Biophys. Acta. 267:275-290.
21. Thayer, W. S., and P. C. Hinkle. 1975. Synthesis of adenosine triphosphate by an artificially
imposed electrochemical proton gradient. J. Biol. Chem. 250:5330-5335.
22. Tinoco, I., K. Sauer, and J. C. Wang. 1978. Physical Chemistry: Principles and Application in
the Biological Sciences. Prentice-Hall, Englewood.
23. West, I., and P. Mitchell. 1972. Proton-coupled β-galactoside translocation in non-metabolizing
Escherichia coli. J. Bioenerg. 3:445-462.

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BTOL-Bioenergetics 1

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Table 1-1. Constants and Conversions

Constant Symbol SI Units Value cgs Units

Avogadro’s number N 6.02 x 1023 mol–1

C. Useful Mixed Constants

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1-2. The First Law of Thermodynamics

1-2.1 Properties of the energy, E:

1-2.2. Work & Heat.

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The pressure-volume work accomplished by expansion (or compression) of a gas at

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1-3. Different Kinds of Work.

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The First Law of Thermodynamics expresses the impossibility of constructing a machine

1-5.1. The approach to equilibrium.

1-6. Entropy and the Second Law of Thermodynamics

The entropy (from Gr., EMBED Equation.3 change, turn) is a state

1-6.1. In the statistical formulation due to Boltzmann,

Copyright by Cramer and Soriano, 2002

dSI > 0 (1-10a)

1-6.1.1 Example of Calculation of Thermodynamic Probability, W:

Calculate the number of combinations, Wj,k of four identical molecules, N = 4, contained in

where N! = N(N – 1)(N – 2) ...(1).

bcd acd abd abc

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1-6.2.2. Statement of the Second Law of Thermodynamics Using the Clausius

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dSI > 0, (1-13a)

dSI > dQ/T (1-14)

1-6.2.3 Brief Mathematical Summary of Second Law of Thermodynamics

I. Isolated system (dS ≥ 0)

dStotal = dS1 + dS2 = -dQ/T1 + dQ/T2 (1-15a)

dStotal > 0, (1-15b)

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Figure 1-3. Two experiments on an isothermal (constant temperature, dE = 0) expansion of a dilute

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Then, as in the discussion of the reversible transition in Fig. 1-1,

since V2=2V1 (Fig. 1-3C).

1-6.4.2. In the second experiment, the partition is removed to allow a redistribution of

From the Boltzmann expression (eqn.1- 7) for S,

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Copyright by Cramer and Soriano, 2002

1-6.5.1. Work Done by System, (I) Isothermal Transitions

and an equal amount of heat, ∆Q1, is released by it.

i. e., VC/VB = VD/VA, and VC/VD = VB/VA (1-18)

Then, the net work done by the system,

∆Scycle = ∆SAB + ∆SBC + ∆SCD + ∆SDA (1-20a)

Copyright by Cramer and Soriano, 2002

∆Scycle = ∆QAB/ T2 + ∆QCD/T1 = nR ln(VB /VA ) - nR ln(VB /VA ) = 0 (1-20c)

1-7. Free Energy.

From the second law, dS ≥ dQ/T, and by substitution,

or defining the Gibbs Free Energy, G, is defined as G ≡ H – TS,

Copyright by Cramer and Soriano, 2002

Copyright by Cramer and Soriano, 2002

Gly Asp Ala

Copyright by Cramer and Soriano, 2002

1-7.2. Concentration Dependence of the Free Energy.