Homework #3 - Mar - 17

CHEM 400 (Spring, 2023)
Homework 3 – Due: Mar 28 (Tuesday), midnight
1. (5 pts) High-spin iron(II) is too large for the opening of the porphyrin ring, but low-spin
iron(II) can be accommodated in the opening. Why does the high-spin ion have a larger radius?
(For simplicity, assume iron has a perfect octahedral geometry with two additional axial ligands)
2. (12 pts) Predict the spin state of the following molecules (e.g., S = 1), where J indicates the
magnetic coupling constant (Ĥ = –2J S1S2). Assume that all the Mn centers are high-spin.
Note: you don’t have to know what ‘tmeda’, ‘bpy’, or ‘bispocene’ are in order to solve this
problem.
(a) MnII2(OH2)(OAc)(tmeda)2 (J = –2.95 cm–1)
(b) [MnIII2(μ-O)(μ-O2CPh)2(N3)2(bpy)2] (J = 8.8 cm–1)
(c) [MnIIIMnIV(μ-O)2(μ-OAc)2]2+ (J = –220 cm–1)
(d) [MnIII2(μ-O)(μ-OAc)(bispocen)2]3+ (J = 19.5 cm–1)
3. (a) (15 pts) Sketch the metal active site structures of the deoxy- and oxy- forms of
hemoglobin, hemocyanin, and hemerythrin. Be sure to include the oxidation states of the metal
centers, the amino acid side chain(s), and the proper heme prosthetic group.
(b) (6 pts) The O-O vibrational data from the aforementioned O2-carrier proteins are 1105
cm–1, 844 cm–1 and 750 cm–1 (not in any specific order). Match the data with the right
protein and explain how your assignments are made.
4. (12 pts) For each of the reactions listed below, give the balanced reaction product(s) and
identification of the enzyme and metal center (e.g., carbonic anhydrase, Zn2+) that catalyzes it:
(a) CH4 + O2 + H+ + NADH ®
O
(b) O
_
+ O2 ®
NH 3+
HO
OH
(c) + O2 ®
OH
(d) SO32– + H2O + cytochrome c (oxidized) ®

5. (10 pts) A resonance Raman spectrum (i.e., vibrational spectrum) of the peroxo intermediate
in a newly identified copper oxidase, which has the formula [Cu2O2]2+, reveals a band at 750
cm–1. To prove the assignment of this band as an O-O stretching mode, you can prepare the 18O
analog. At what frequency would you expect the isotopically substituted intermediate to appear?
Hint: The frequency of the stretching vibration of a diatomic molecule, X–Y, is described by
Hooke’s law
! $
𝜈 = "# $%
! ! !
μ = reduced mass, where % = & + & (𝑚' and 𝑚( are the masses of X and Y)
! "
k = force constant
The frequency of the oscillation in units of reciprocal distance (cm–1) can be obtained by
using the relation λν = c, where c is the speed of light, 3 x 1010 cm/sec.
6. (16 pts) As discussed in class Cytochrome P450s catalyze a very broad range of reactions
including some that play an essential role in the production of many steroid hormones including
estrogen. A single enzyme called ‘Aromatase’ catalyzes the conversion of the C19 methyl group
of 1 to alcohol (2), which is then converted to an aldehyde intermediate (3) and ultimately to
estrogen (4) and formate. The first two steps are thought to be similar to those discussed in class
that is catalyzed by Camphor P450. The third step is thought to involve a different reactive iron
intermediate. During these reactions catalyzed by a single enzyme, three molecules of NADPH
are oxidized to NADP+ and three molecules of O2 are consumed.
The following information has been obtained about aromatase. In the conversion of 1 to 2,
labeling experiments reveal that the oxygen in 2 is derived from O2. In the second step of the
reaction involving conversion of 2 to 3, labeling studies have shown that the aldehyde oxygen of
3 is derived solely from the first mole of O2 consumed. In the final product 4, labeling studies
have revealed that oxygen atoms are derived from the first and third moles of O2 consumed.
A recent structure of microsomal cytochrome P450 and primary sequence alignments gave a
model of the active site of aromatase shown in Figure 1. This model was able to account for
some of the mechanistic studies carried out on this enzyme.
Figure 1: A model of aromatase based on a structure of a human microsomal P450. The structure of substrate 1 is
modeled into the active site with the carbonyl oxygen shown in red and the C-19 methyl pointed up.
Observations:
– Mutation of D309 to an alanine prohibited this reaction, while the enzyme still
was able to catalyze 1 to 2 and 2 to 3. The reaction of 3 to 4 was completely
inhibited.
– A mutation of T310 to alanine was examined and shown to inhibit the conversion
1 to 2 and 2 to 3. Instead, H2O2 was detected. However, this same mutant allowed
the conversion of 3 to 4.
– When [2-3H]-1 (i.e., compound 1 whose protons at the 2-position are substituted
with tritium) was incubated with aromatase, a small amount of 3H2O was released.
Questions:
1. Propose a mechanism to accommodate the first two steps in the reaction (1 to 2 and 2
to 3) based on the supposition that the mechanism is similar to Camphor cytochrome
P450.
2. Propose a mechanism for the conversion of 3 to 4. Use the information provided by
the site directed mutagenesis experiments to help you propose a mechanism for this
step. Clearly describe how this data is accommodated by your proposal.

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CHEM 400 (Spring, 2023)

Homework 3 – Due: Mar 28 (Tuesday), midnight

(a) MnII2(OH2)(OAc)(tmeda)2 (J = –2.95 cm–1)

(b) [MnIII2(μ-O)(μ-O2CPh)2(N3)2(bpy)2] (J = 8.8 cm–1)

(c) [MnIIIMnIV(μ-O)2(μ-OAc)2]2+ (J = –220 cm–1)

(d) [MnIII2(μ-O)(μ-OAc)(bispocen)2]3+ (J = 19.5 cm–1)

(a) CH4 + O2 + H+ + NADH ®

(d) SO32– + H2O + cytochrome c (oxidized) ®

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