Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It ... more Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes. The turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized to the metastable OH state, and a reductive phase, in which OH is reduced back to the R state. During each phase, two protons are translocated across the membrane. However, if OH is allowed to relax to the resting oxidized state (O), a redox equivalent to OH, its subsequent reduction to R is incapable of driving proton translocation. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX), we show that the heme a3 iron and CuB in the active site of the O state, like those in the OH state, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, critical for the oxyg...
bioRxiv (Cold Spring Harbor Laboratory), Mar 22, 2023
Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It ... more Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes, thereby establishing the proton gradient required for ATP synthesis 1. The full turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized by molecular oxygen to the metastable oxidized OH state, and a reductive phase, in which OH is reduced back to the R state. During each of the two phases, two protons are translocated across the membranes 2. However, if OH is allowed to relax to the resting oxidized state (O), a redox equivalent to OH, its subsequent reduction to R is incapable of driving proton translocation 2,3. How the O state structurally differs from OH remains an enigma in modern bioenergetics. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX) 4 , we show that the heme a3 iron and CuB in the active site of the O state, like those in the OH state 5,6 , are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, a residue covalently linked to one of the three CuB ligands and critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from OH, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide new insights into the proton translocation mechanism of CcO. Main Mammalian CcO is a large integral membrane protein comprised of 13 subunits. It contains four redox active centers, CuA, heme a, and a heme a3/CuB binuclear center (BNC) (Fig. 1A). Molecular oxygen binds to the heme a3 iron in the BNC, where it is reduced to two water molecules by accepting four electrons from cytochrome c and four protons (the "substrate" protons) from the negative side (N-side) of the mitochondrial membrane (Fig. 1A). The energy derived from the oxygen reduction chemistry is used to drive the translocation of four protons (the "pumped" protons) from the N-side to the positive side (P-side) of the membrane 1,7. Strong evidence suggests that the substrate protons are delivered to the BNC via the D and K-channel (see Extended Data
The conversion of light into usable chemical and mechanical energy is pivotal to several biologic... more The conversion of light into usable chemical and mechanical energy is pivotal to several biological and chemical processes, many of which occur in solution. To understand the structure-function relationships mediating these processes, a technique with high spatial and temporal resolutions is required. Here, we report on the design and commissioning of a liquid-phase mega-electron-volt (MeV) ultrafast electron diffraction instrument for the study of structural dynamics in solution. Limitations posed by the shallow penetration depth of electrons and the resulting information loss due to multiple scattering and the technical challenge of delivering liquids to vacuum were overcome through the use of MeV electrons and a gas-accelerated thin liquid sheet jet. To demonstrate the capabilities of this instrument, the structure of water and its network were resolved up to the 3rd hydration shell with a spatial resolution of 0.6 Å ; preliminary time-resolved experiments demonstrated a temporal resolution of 200 fs.
A 3D-printed modular droplet injector successfully delivered microcrystals of human NAD(P)H:quino... more A 3D-printed modular droplet injector successfully delivered microcrystals of human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin with electrical stimulation in a serial crystallography experiment at 120 Hz repetition rate.
Beamline 10.3.2 at the ALS is a bend-magnet line designed mostly for work on environmental proble... more Beamline 10.3.2 at the ALS is a bend-magnet line designed mostly for work on environmental problems involving heavy-metal speciation and location. It offers a unique combination of X-ray¯uorescence mapping, X-ray microspectroscopy and micro-X-ray diffraction. The optics allow the user to trade spot size for¯ux in a size range of 5±17 mm in an energy range of 3±17 keV. The focusing uses a Kirkpatrick±Baez mirror pair to image a variable-size virtual source onto the sample. Thus, the user can reduce the effective size of the source, thereby reducing the spot size on the sample, at the cost of ux. This decoupling from the actual source also allows for some independence from source motion. The X-ray¯uorescence mapping is performed with a continuously scanning stage which avoids the time overhead incurred by step-and-repeat mapping schemes. The special features of this beamline are described, and some scienti®c results shown.
X-ray microprobes are among the most important new analytical techniques, which have emerged from... more X-ray microprobes are among the most important new analytical techniques, which have emerged from 3 rd generation synchrotron facilities. Here we show how X-ray fluorescence, diffraction, and absorption can be used in parallel to determine the structural form of trace elements in heterogeneous matrices at the micrometer-scale of resolution. Scanning X-ray microfluorescence (µSXRF) and microdiffraction (µSXRD) first are used to identify the host solid phase by mapping the distributions of elements and solid species, respectively. Micro-extended X-ray absorption fine structure (µEXAFS) spectroscopy is then used to determine the mechanism of trace element binding by the host phase at the molecular scale. To illustrate the complementary application of these three techniques, we studied how nickel is sequestered in soil ferromanganese nodules, an overwhelmingly complex natural matrix consisting of submicrometer to nanometer sized particles with varying structures and chemical compositions. We show that nickel substitutes for Mn 3+ in the manganese layer of the MnO 2 -Al(OH) 3 mixed-layer oxide lithiophorite. The affinity of Ni for lithiophorite was characteristic of micronodules sampled from soils across the USA and Europe.
Beamline 10.3.2 at the ALS is a bend-magnet line designed mostly for work on environmental proble... more Beamline 10.3.2 at the ALS is a bend-magnet line designed mostly for work on environmental problems involving heavy-metal speciation and location. It offers a unique combination of X-ray¯uorescence mapping, X-ray microspectroscopy and micro-X-ray diffraction. The optics allow the user to trade spot size for¯ux in a size range of 5±17 mm in an energy range of 3±17 keV. The focusing uses a Kirkpatrick±Baez mirror pair to image a variable-size virtual source onto the sample. Thus, the user can reduce the effective size of the source, thereby reducing the spot size on the sample, at the cost of ux. This decoupling from the actual source also allows for some independence from source motion. The X-ray¯uorescence mapping is performed with a continuously scanning stage which avoids the time overhead incurred by step-and-repeat mapping schemes. The special features of this beamline are described, and some scienti®c results shown.
Beamline 10.3.2 at the ALS is a bend-magnet line designed mostly for work on environmental proble... more Beamline 10.3.2 at the ALS is a bend-magnet line designed mostly for work on environmental problems involving heavy-metal speciation and location. It offers a unique combination of X-ray¯uorescence mapping, X-ray microspectroscopy and micro-X-ray diffraction. The optics allow the user to trade spot size for¯ux in a size range of 5±17 mm in an energy range of 3±17 keV. The focusing uses a Kirkpatrick±Baez mirror pair to image a variable-size virtual source onto the sample. Thus, the user can reduce the effective size of the source, thereby reducing the spot size on the sample, at the cost of ux. This decoupling from the actual source also allows for some independence from source motion. The X-ray¯uorescence mapping is performed with a continuously scanning stage which avoids the time overhead incurred by step-and-repeat mapping schemes. The special features of this beamline are described, and some scienti®c results shown.
Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It ... more Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes. The turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized to the metastable OH state, and a reductive phase, in which OH is reduced back to the R state. During each phase, two protons are translocated across the membrane. However, if OH is allowed to relax to the resting oxidized state (O), a redox equivalent to OH, its subsequent reduction to R is incapable of driving proton translocation. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX), we show that the heme a3 iron and CuB in the active site of the O state, like those in the OH state, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, critical for the oxyg...
bioRxiv (Cold Spring Harbor Laboratory), Mar 22, 2023
Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It ... more Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes, thereby establishing the proton gradient required for ATP synthesis 1. The full turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized by molecular oxygen to the metastable oxidized OH state, and a reductive phase, in which OH is reduced back to the R state. During each of the two phases, two protons are translocated across the membranes 2. However, if OH is allowed to relax to the resting oxidized state (O), a redox equivalent to OH, its subsequent reduction to R is incapable of driving proton translocation 2,3. How the O state structurally differs from OH remains an enigma in modern bioenergetics. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX) 4 , we show that the heme a3 iron and CuB in the active site of the O state, like those in the OH state 5,6 , are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, a residue covalently linked to one of the three CuB ligands and critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from OH, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide new insights into the proton translocation mechanism of CcO. Main Mammalian CcO is a large integral membrane protein comprised of 13 subunits. It contains four redox active centers, CuA, heme a, and a heme a3/CuB binuclear center (BNC) (Fig. 1A). Molecular oxygen binds to the heme a3 iron in the BNC, where it is reduced to two water molecules by accepting four electrons from cytochrome c and four protons (the "substrate" protons) from the negative side (N-side) of the mitochondrial membrane (Fig. 1A). The energy derived from the oxygen reduction chemistry is used to drive the translocation of four protons (the "pumped" protons) from the N-side to the positive side (P-side) of the membrane 1,7. Strong evidence suggests that the substrate protons are delivered to the BNC via the D and K-channel (see Extended Data
The conversion of light into usable chemical and mechanical energy is pivotal to several biologic... more The conversion of light into usable chemical and mechanical energy is pivotal to several biological and chemical processes, many of which occur in solution. To understand the structure-function relationships mediating these processes, a technique with high spatial and temporal resolutions is required. Here, we report on the design and commissioning of a liquid-phase mega-electron-volt (MeV) ultrafast electron diffraction instrument for the study of structural dynamics in solution. Limitations posed by the shallow penetration depth of electrons and the resulting information loss due to multiple scattering and the technical challenge of delivering liquids to vacuum were overcome through the use of MeV electrons and a gas-accelerated thin liquid sheet jet. To demonstrate the capabilities of this instrument, the structure of water and its network were resolved up to the 3rd hydration shell with a spatial resolution of 0.6 Å ; preliminary time-resolved experiments demonstrated a temporal resolution of 200 fs.
A 3D-printed modular droplet injector successfully delivered microcrystals of human NAD(P)H:quino... more A 3D-printed modular droplet injector successfully delivered microcrystals of human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin with electrical stimulation in a serial crystallography experiment at 120 Hz repetition rate.
Beamline 10.3.2 at the ALS is a bend-magnet line designed mostly for work on environmental proble... more Beamline 10.3.2 at the ALS is a bend-magnet line designed mostly for work on environmental problems involving heavy-metal speciation and location. It offers a unique combination of X-ray¯uorescence mapping, X-ray microspectroscopy and micro-X-ray diffraction. The optics allow the user to trade spot size for¯ux in a size range of 5±17 mm in an energy range of 3±17 keV. The focusing uses a Kirkpatrick±Baez mirror pair to image a variable-size virtual source onto the sample. Thus, the user can reduce the effective size of the source, thereby reducing the spot size on the sample, at the cost of ux. This decoupling from the actual source also allows for some independence from source motion. The X-ray¯uorescence mapping is performed with a continuously scanning stage which avoids the time overhead incurred by step-and-repeat mapping schemes. The special features of this beamline are described, and some scienti®c results shown.
X-ray microprobes are among the most important new analytical techniques, which have emerged from... more X-ray microprobes are among the most important new analytical techniques, which have emerged from 3 rd generation synchrotron facilities. Here we show how X-ray fluorescence, diffraction, and absorption can be used in parallel to determine the structural form of trace elements in heterogeneous matrices at the micrometer-scale of resolution. Scanning X-ray microfluorescence (µSXRF) and microdiffraction (µSXRD) first are used to identify the host solid phase by mapping the distributions of elements and solid species, respectively. Micro-extended X-ray absorption fine structure (µEXAFS) spectroscopy is then used to determine the mechanism of trace element binding by the host phase at the molecular scale. To illustrate the complementary application of these three techniques, we studied how nickel is sequestered in soil ferromanganese nodules, an overwhelmingly complex natural matrix consisting of submicrometer to nanometer sized particles with varying structures and chemical compositions. We show that nickel substitutes for Mn 3+ in the manganese layer of the MnO 2 -Al(OH) 3 mixed-layer oxide lithiophorite. The affinity of Ni for lithiophorite was characteristic of micronodules sampled from soils across the USA and Europe.
Beamline 10.3.2 at the ALS is a bend-magnet line designed mostly for work on environmental proble... more Beamline 10.3.2 at the ALS is a bend-magnet line designed mostly for work on environmental problems involving heavy-metal speciation and location. It offers a unique combination of X-ray¯uorescence mapping, X-ray microspectroscopy and micro-X-ray diffraction. The optics allow the user to trade spot size for¯ux in a size range of 5±17 mm in an energy range of 3±17 keV. The focusing uses a Kirkpatrick±Baez mirror pair to image a variable-size virtual source onto the sample. Thus, the user can reduce the effective size of the source, thereby reducing the spot size on the sample, at the cost of ux. This decoupling from the actual source also allows for some independence from source motion. The X-ray¯uorescence mapping is performed with a continuously scanning stage which avoids the time overhead incurred by step-and-repeat mapping schemes. The special features of this beamline are described, and some scienti®c results shown.
Beamline 10.3.2 at the ALS is a bend-magnet line designed mostly for work on environmental proble... more Beamline 10.3.2 at the ALS is a bend-magnet line designed mostly for work on environmental problems involving heavy-metal speciation and location. It offers a unique combination of X-ray¯uorescence mapping, X-ray microspectroscopy and micro-X-ray diffraction. The optics allow the user to trade spot size for¯ux in a size range of 5±17 mm in an energy range of 3±17 keV. The focusing uses a Kirkpatrick±Baez mirror pair to image a variable-size virtual source onto the sample. Thus, the user can reduce the effective size of the source, thereby reducing the spot size on the sample, at the cost of ux. This decoupling from the actual source also allows for some independence from source motion. The X-ray¯uorescence mapping is performed with a continuously scanning stage which avoids the time overhead incurred by step-and-repeat mapping schemes. The special features of this beamline are described, and some scienti®c results shown.
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