UC Santa Cruz

Postdoctoral scholar

Theoretical and computational biophysics research; classical, quantum, and combined classical/quantum simulations of terminal heme-copper oxidases.
Parameterized CHARMM force field for transition metal-containing enzyme active site models
Developed scripts to manage file systems for large data sets
Developed scripts to automate running multiple calculations efficiently

UC Santa Cruz

Lecturer

Course instructor for Chemistry 1P: Chemistry Essentials. The course introduces students to basic chemistry concepts including atoms, molecules, isotopes, atomic and molecular mass, the mole, chemical equations and reactions, and stoichiometry.

Cabrillo College

Adjunct Professor of Chemistry

William worked at Cabrillo College as a Adjunct Professor of Chemistry

Monterey Peninsula College

Adjunct Professor of Chemistry

William worked at Monterey Peninsula College as a Adjunct Professor of Chemistry

Ashwin-Ushas Corporation Inc.

Senior Scientist, Computational Chemistry

Computational biophysics studies of dioxygen reduction in cytochrome P450s
Managed computational chemistry laboratory including hardware acquisition and maintenance, software installation, and resource scheduling

Spectral Identification of Intermediates Generated during the Reaction of Dioxygen with the Wild-type and EQ(I-286) Mutant of Rhodobacter Sphaeroides Cytochrome c Oxidase

Biochemistry 51(46) 9302–9311

Spectral Identification of Intermediates Generated during the Reaction of Dioxygen with the Wild-type and EQ(I-286) Mutant of Rhodobacter Sphaeroides Cytochrome c Oxidase

Biochemistry 51(46) 9302–9311

Conserved Glycine 232 in the Ligand Channel of ba3 Cytochrome Oxidase from Thermus thermophilus

Biochemistry 53(27), 4467—4475

Knowing how the protein environment modulates ligand pathways and redox centers in the respiratory heme-copper oxidases is fundamental for understanding the relationship between the structure and function of these enzymes. In this study, we investigated the reactions of O2 and NO with the fully reduced G232V mutant of ba3 cytochrome c oxidase from Thermus thermophilus (Tt ba3) in which a conserved glycine residue in the O2 channel of the enzyme was replaced with a bulkier valine residue. Previous studies of the homologous mutant of Rhodobacter sphaeroides aa3 cytochrome c oxidase suggested that the valine completely blocked the access of O2 to the active site [Salomonsson, L., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 11617−11621]. Using photolabile O2 and NO carriers, we find by using time-resolved optical absorption spectroscopy that the rates of O2 and NO binding are not significantly affected in the Tt ba3 G232V mutant. Classical molecular dynamics simulations of diffusion of O2 to the active site in the wild-type enzyme and G232V mutant show that the insertion of the larger valine residue in place of the glycine appears to open up other O2 and NO exit/entrance pathways that allow these ligands unhindered access to the active site, thus compensating for the larger valine residue.

Spectral Identification of Intermediates Generated during the Reaction of Dioxygen with the Wild-type and EQ(I-286) Mutant of Rhodobacter Sphaeroides Cytochrome c Oxidase

Biochemistry 51(46) 9302–9311

Conserved Glycine 232 in the Ligand Channel of ba3 Cytochrome Oxidase from Thermus thermophilus

Biochemistry 53(27), 4467—4475

Knowing how the protein environment modulates ligand pathways and redox centers in the respiratory heme-copper oxidases is fundamental for understanding the relationship between the structure and function of these enzymes. In this study, we investigated the reactions of O2 and NO with the fully reduced G232V mutant of ba3 cytochrome c oxidase from Thermus thermophilus (Tt ba3) in which a conserved glycine residue in the O2 channel of the enzyme was replaced with a bulkier valine residue. Previous studies of the homologous mutant of Rhodobacter sphaeroides aa3 cytochrome c oxidase suggested that the valine completely blocked the access of O2 to the active site [Salomonsson, L., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 11617−11621]. Using photolabile O2 and NO carriers, we find by using time-resolved optical absorption spectroscopy that the rates of O2 and NO binding are not significantly affected in the Tt ba3 G232V mutant. Classical molecular dynamics simulations of diffusion of O2 to the active site in the wild-type enzyme and G232V mutant show that the insertion of the larger valine residue in place of the glycine appears to open up other O2 and NO exit/entrance pathways that allow these ligands unhindered access to the active site, thus compensating for the larger valine residue.

Ligand Access to the Active Site in Thermus thermophilus ba3 and Bovine Heart aa3 Cytochrome Oxidases.

Biochemistry 52, 640–652

Knowledge of the structure and dynamics of the ligand channel(s) in heme-copper oxidases is critical for understanding how the protein environment modulates the functions of these enzymes. Using photolabile NO and O2 carriers, we recently found that NO and O2 binding in Thermus thermophilus (Tt ) ba3 is ∼10 times faster than in the bovine enzyme, indicating that inherent structural differences affect ligand access in these enzymes. Using X-ray crystallog- raphy, time-resolved optical absorption measurements, and theoretical calculations, we investigated ligand access in wild- type Tt ba3 and the mutants, Y133W, T231F, and Y133W/T231F, in which tyrosine and threonine in the O2 channel of Tt ba3 are replaced by the corresponding bulkier tryptophan and phenylalanine, respectively, present in the aa3 enzymes. NO binding in Y133W and Y133W/T231F was found to be 5 times slower than in wild-type ba3 and the T231F mutant. The results show that the Tt ba3 Y133W mutation and the bovine W126 residue physically impede NO access to the binuclear center. In the bovine enzyme, there is a hydrophobic “way station”, which may further slow ligand access to the active site. Classical simulations of diffusion of Xe to the active sites in ba3 and bovine aa3 show conformational freedom of the bovine F238 and the F231 side chain of the Tt ba3 Y133W/T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme.

Spectral Identification of Intermediates Generated during the Reaction of Dioxygen with the Wild-type and EQ(I-286) Mutant of Rhodobacter Sphaeroides Cytochrome c Oxidase

Biochemistry 51(46) 9302–9311

Conserved Glycine 232 in the Ligand Channel of ba3 Cytochrome Oxidase from Thermus thermophilus

Biochemistry 53(27), 4467—4475

Knowing how the protein environment modulates ligand pathways and redox centers in the respiratory heme-copper oxidases is fundamental for understanding the relationship between the structure and function of these enzymes. In this study, we investigated the reactions of O2 and NO with the fully reduced G232V mutant of ba3 cytochrome c oxidase from Thermus thermophilus (Tt ba3) in which a conserved glycine residue in the O2 channel of the enzyme was replaced with a bulkier valine residue. Previous studies of the homologous mutant of Rhodobacter sphaeroides aa3 cytochrome c oxidase suggested that the valine completely blocked the access of O2 to the active site [Salomonsson, L., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 11617−11621]. Using photolabile O2 and NO carriers, we find by using time-resolved optical absorption spectroscopy that the rates of O2 and NO binding are not significantly affected in the Tt ba3 G232V mutant. Classical molecular dynamics simulations of diffusion of O2 to the active site in the wild-type enzyme and G232V mutant show that the insertion of the larger valine residue in place of the glycine appears to open up other O2 and NO exit/entrance pathways that allow these ligands unhindered access to the active site, thus compensating for the larger valine residue.

Ligand Access to the Active Site in Thermus thermophilus ba3 and Bovine Heart aa3 Cytochrome Oxidases.

Biochemistry 52, 640–652

Knowledge of the structure and dynamics of the ligand channel(s) in heme-copper oxidases is critical for understanding how the protein environment modulates the functions of these enzymes. Using photolabile NO and O2 carriers, we recently found that NO and O2 binding in Thermus thermophilus (Tt ) ba3 is ∼10 times faster than in the bovine enzyme, indicating that inherent structural differences affect ligand access in these enzymes. Using X-ray crystallog- raphy, time-resolved optical absorption measurements, and theoretical calculations, we investigated ligand access in wild- type Tt ba3 and the mutants, Y133W, T231F, and Y133W/T231F, in which tyrosine and threonine in the O2 channel of Tt ba3 are replaced by the corresponding bulkier tryptophan and phenylalanine, respectively, present in the aa3 enzymes. NO binding in Y133W and Y133W/T231F was found to be 5 times slower than in wild-type ba3 and the T231F mutant. The results show that the Tt ba3 Y133W mutation and the bovine W126 residue physically impede NO access to the binuclear center. In the bovine enzyme, there is a hydrophobic “way station”, which may further slow ligand access to the active site. Classical simulations of diffusion of Xe to the active sites in ba3 and bovine aa3 show conformational freedom of the bovine F238 and the F231 side chain of the Tt ba3 Y133W/T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme.

The CO Photodissociation and Recombination Dynamics of the W172Y/ F282T Ligand Channel Mutant of Rhodobacter sphaeroides aa3 Cytochrome c Oxidase

Photochemistry and Photobiology

In the ligand channel of the cytochrome c oxidase from Rhodobacter sphaeroides (Rs aa3) W172 and F282 have been proposed to generate a constriction that may slow ligand access to and from the active site. To explore this issue, the tryptophan and phenylalanine residues in Rs aa3 were mutated to the less bulky tyrosine and threonine residues, respectively, which occupy these sites in Thermus thermophilus (Tt) ba3 cytochrome oxidase. The CO photolysis and recombination dynamics of the reduced wild-type Rs aa3 and the W172Y/F282T mutant were investigated using time-resolved optical absorption spectroscopy. The spectral changes associated with the multiple processes are attributed to different conformers. The major CO recombination process (44 μs) in the W172Y/F282T mutant is ~500 times faster than the predominant CO recombination process in the wild-type enzyme (~23 ms). Classical dynamic simulations of the wild-type enzyme and double mutant showed significant structural changes at the active site in the mutant, including movement of the heme a3 ring-D propionate toward CuB and reduced binuclear center cavity volume. These structural changes effectively close the ligand exit pathway from the binuclear center, providing a basis for the faster CO recombination in the double mutant.

Spectral Identification of Intermediates Generated during the Reaction of Dioxygen with the Wild-type and EQ(I-286) Mutant of Rhodobacter Sphaeroides Cytochrome c Oxidase

Biochemistry 51(46) 9302–9311

Conserved Glycine 232 in the Ligand Channel of ba3 Cytochrome Oxidase from Thermus thermophilus

Biochemistry 53(27), 4467—4475

Knowing how the protein environment modulates ligand pathways and redox centers in the respiratory heme-copper oxidases is fundamental for understanding the relationship between the structure and function of these enzymes. In this study, we investigated the reactions of O2 and NO with the fully reduced G232V mutant of ba3 cytochrome c oxidase from Thermus thermophilus (Tt ba3) in which a conserved glycine residue in the O2 channel of the enzyme was replaced with a bulkier valine residue. Previous studies of the homologous mutant of Rhodobacter sphaeroides aa3 cytochrome c oxidase suggested that the valine completely blocked the access of O2 to the active site [Salomonsson, L., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 11617−11621]. Using photolabile O2 and NO carriers, we find by using time-resolved optical absorption spectroscopy that the rates of O2 and NO binding are not significantly affected in the Tt ba3 G232V mutant. Classical molecular dynamics simulations of diffusion of O2 to the active site in the wild-type enzyme and G232V mutant show that the insertion of the larger valine residue in place of the glycine appears to open up other O2 and NO exit/entrance pathways that allow these ligands unhindered access to the active site, thus compensating for the larger valine residue.

Ligand Access to the Active Site in Thermus thermophilus ba3 and Bovine Heart aa3 Cytochrome Oxidases.

Biochemistry 52, 640–652

Knowledge of the structure and dynamics of the ligand channel(s) in heme-copper oxidases is critical for understanding how the protein environment modulates the functions of these enzymes. Using photolabile NO and O2 carriers, we recently found that NO and O2 binding in Thermus thermophilus (Tt ) ba3 is ∼10 times faster than in the bovine enzyme, indicating that inherent structural differences affect ligand access in these enzymes. Using X-ray crystallog- raphy, time-resolved optical absorption measurements, and theoretical calculations, we investigated ligand access in wild- type Tt ba3 and the mutants, Y133W, T231F, and Y133W/T231F, in which tyrosine and threonine in the O2 channel of Tt ba3 are replaced by the corresponding bulkier tryptophan and phenylalanine, respectively, present in the aa3 enzymes. NO binding in Y133W and Y133W/T231F was found to be 5 times slower than in wild-type ba3 and the T231F mutant. The results show that the Tt ba3 Y133W mutation and the bovine W126 residue physically impede NO access to the binuclear center. In the bovine enzyme, there is a hydrophobic “way station”, which may further slow ligand access to the active site. Classical simulations of diffusion of Xe to the active sites in ba3 and bovine aa3 show conformational freedom of the bovine F238 and the F231 side chain of the Tt ba3 Y133W/T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme.

The CO Photodissociation and Recombination Dynamics of the W172Y/ F282T Ligand Channel Mutant of Rhodobacter sphaeroides aa3 Cytochrome c Oxidase

Photochemistry and Photobiology

In the ligand channel of the cytochrome c oxidase from Rhodobacter sphaeroides (Rs aa3) W172 and F282 have been proposed to generate a constriction that may slow ligand access to and from the active site. To explore this issue, the tryptophan and phenylalanine residues in Rs aa3 were mutated to the less bulky tyrosine and threonine residues, respectively, which occupy these sites in Thermus thermophilus (Tt) ba3 cytochrome oxidase. The CO photolysis and recombination dynamics of the reduced wild-type Rs aa3 and the W172Y/F282T mutant were investigated using time-resolved optical absorption spectroscopy. The spectral changes associated with the multiple processes are attributed to different conformers. The major CO recombination process (44 μs) in the W172Y/F282T mutant is ~500 times faster than the predominant CO recombination process in the wild-type enzyme (~23 ms). Classical dynamic simulations of the wild-type enzyme and double mutant showed significant structural changes at the active site in the mutant, including movement of the heme a3 ring-D propionate toward CuB and reduced binuclear center cavity volume. These structural changes effectively close the ligand exit pathway from the binuclear center, providing a basis for the faster CO recombination in the double mutant.

Kinetics and Intermediates of the Reaction of Fully Reduced Escherichia coli bo3 Ubiquinol Oxidase with O2

Biochemistry 53(33), 5393—5404

Cytochrome bo3 ubiquinol oxidase from Escherichia coli catalyzes the reduction of O2 to water by ubiquinol. The reaction mechanism and the role of ubiquinol continue to be a subject of discussion. In this study, we report a detailed kinetic scheme of the reaction of cytochrome bo3 with O2 with steps specific to ubiquinol. The reaction was investigated using the CO flow-flash method, and time-resolved optical absorption difference spectra were collected from 1 μs to 20 ms after photolysis. Singular value decomposition-based global exponential fitting resolved five apparent lifetimes, 22 μs, 30 μs, 42 μs, 470 μs, and 2.0 ms. The reaction mechanism was derived by an algebraic kinetic analysis method using frequency-shifted spectra of known bovine states to identify the bo3 intermediates. It shows 42 μs O2 binding (3.8 × 107 M−1 s−1), producing compound A, followed by faster (22 μs) heme b oxidation, yielding a mixture of PR and F, and rapid heme b rereduction by ubiquinol (30 μs), producing the F intermediate and semiquinone. In the 470 μs step, the o3 F state is converted into the o33+ oxidized state, presumably by semiquinone/ubiquinol, without the concomitant oxidation of heme b. The final 2 ms step shows heme b reoxidation and the partial rereduction of the binuclear center and, following O2 binding, the formation of a mixture of P and F during a second turnover cycle. The results show that ubiquinol/semiquinone plays a complex role in the mechanism of O2 reduction by bo3, displaying kinetic steps that have no analogy in the CuA-containing heme-copper oxidases.

Spectral Identification of Intermediates Generated during the Reaction of Dioxygen with the Wild-type and EQ(I-286) Mutant of Rhodobacter Sphaeroides Cytochrome c Oxidase

Biochemistry 51(46) 9302–9311

Conserved Glycine 232 in the Ligand Channel of ba3 Cytochrome Oxidase from Thermus thermophilus

Biochemistry 53(27), 4467—4475

Knowing how the protein environment modulates ligand pathways and redox centers in the respiratory heme-copper oxidases is fundamental for understanding the relationship between the structure and function of these enzymes. In this study, we investigated the reactions of O2 and NO with the fully reduced G232V mutant of ba3 cytochrome c oxidase from Thermus thermophilus (Tt ba3) in which a conserved glycine residue in the O2 channel of the enzyme was replaced with a bulkier valine residue. Previous studies of the homologous mutant of Rhodobacter sphaeroides aa3 cytochrome c oxidase suggested that the valine completely blocked the access of O2 to the active site [Salomonsson, L., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 11617−11621]. Using photolabile O2 and NO carriers, we find by using time-resolved optical absorption spectroscopy that the rates of O2 and NO binding are not significantly affected in the Tt ba3 G232V mutant. Classical molecular dynamics simulations of diffusion of O2 to the active site in the wild-type enzyme and G232V mutant show that the insertion of the larger valine residue in place of the glycine appears to open up other O2 and NO exit/entrance pathways that allow these ligands unhindered access to the active site, thus compensating for the larger valine residue.

Ligand Access to the Active Site in Thermus thermophilus ba3 and Bovine Heart aa3 Cytochrome Oxidases.

Biochemistry 52, 640–652

Knowledge of the structure and dynamics of the ligand channel(s) in heme-copper oxidases is critical for understanding how the protein environment modulates the functions of these enzymes. Using photolabile NO and O2 carriers, we recently found that NO and O2 binding in Thermus thermophilus (Tt ) ba3 is ∼10 times faster than in the bovine enzyme, indicating that inherent structural differences affect ligand access in these enzymes. Using X-ray crystallog- raphy, time-resolved optical absorption measurements, and theoretical calculations, we investigated ligand access in wild- type Tt ba3 and the mutants, Y133W, T231F, and Y133W/T231F, in which tyrosine and threonine in the O2 channel of Tt ba3 are replaced by the corresponding bulkier tryptophan and phenylalanine, respectively, present in the aa3 enzymes. NO binding in Y133W and Y133W/T231F was found to be 5 times slower than in wild-type ba3 and the T231F mutant. The results show that the Tt ba3 Y133W mutation and the bovine W126 residue physically impede NO access to the binuclear center. In the bovine enzyme, there is a hydrophobic “way station”, which may further slow ligand access to the active site. Classical simulations of diffusion of Xe to the active sites in ba3 and bovine aa3 show conformational freedom of the bovine F238 and the F231 side chain of the Tt ba3 Y133W/T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme.

The CO Photodissociation and Recombination Dynamics of the W172Y/ F282T Ligand Channel Mutant of Rhodobacter sphaeroides aa3 Cytochrome c Oxidase

Photochemistry and Photobiology

In the ligand channel of the cytochrome c oxidase from Rhodobacter sphaeroides (Rs aa3) W172 and F282 have been proposed to generate a constriction that may slow ligand access to and from the active site. To explore this issue, the tryptophan and phenylalanine residues in Rs aa3 were mutated to the less bulky tyrosine and threonine residues, respectively, which occupy these sites in Thermus thermophilus (Tt) ba3 cytochrome oxidase. The CO photolysis and recombination dynamics of the reduced wild-type Rs aa3 and the W172Y/F282T mutant were investigated using time-resolved optical absorption spectroscopy. The spectral changes associated with the multiple processes are attributed to different conformers. The major CO recombination process (44 μs) in the W172Y/F282T mutant is ~500 times faster than the predominant CO recombination process in the wild-type enzyme (~23 ms). Classical dynamic simulations of the wild-type enzyme and double mutant showed significant structural changes at the active site in the mutant, including movement of the heme a3 ring-D propionate toward CuB and reduced binuclear center cavity volume. These structural changes effectively close the ligand exit pathway from the binuclear center, providing a basis for the faster CO recombination in the double mutant.

Kinetics and Intermediates of the Reaction of Fully Reduced Escherichia coli bo3 Ubiquinol Oxidase with O2

Biochemistry 53(33), 5393—5404

Cytochrome bo3 ubiquinol oxidase from Escherichia coli catalyzes the reduction of O2 to water by ubiquinol. The reaction mechanism and the role of ubiquinol continue to be a subject of discussion. In this study, we report a detailed kinetic scheme of the reaction of cytochrome bo3 with O2 with steps specific to ubiquinol. The reaction was investigated using the CO flow-flash method, and time-resolved optical absorption difference spectra were collected from 1 μs to 20 ms after photolysis. Singular value decomposition-based global exponential fitting resolved five apparent lifetimes, 22 μs, 30 μs, 42 μs, 470 μs, and 2.0 ms. The reaction mechanism was derived by an algebraic kinetic analysis method using frequency-shifted spectra of known bovine states to identify the bo3 intermediates. It shows 42 μs O2 binding (3.8 × 107 M−1 s−1), producing compound A, followed by faster (22 μs) heme b oxidation, yielding a mixture of PR and F, and rapid heme b rereduction by ubiquinol (30 μs), producing the F intermediate and semiquinone. In the 470 μs step, the o3 F state is converted into the o33+ oxidized state, presumably by semiquinone/ubiquinol, without the concomitant oxidation of heme b. The final 2 ms step shows heme b reoxidation and the partial rereduction of the binuclear center and, following O2 binding, the formation of a mixture of P and F during a second turnover cycle. The results show that ubiquinol/semiquinone plays a complex role in the mechanism of O2 reduction by bo3, displaying kinetic steps that have no analogy in the CuA-containing heme-copper oxidases.

Role of the Conserved Valine 236 in Access of Ligands to the Active Site of Thermus thermophilus ba3 Cytochrome Oxidase

Biochemistry

Knowledge of the role of conserved residues in the ligand channel of heme-copper oxidases is critical for understanding how the protein scaffold modulates the function of these enzymes. In this study, we investigated the role of the conserved valine 236 in the ligand channel of ba3 cytochrome c oxidase from Thermus thermophilus by mutating the residue to a more polar (V236T), smaller (V236A), or larger (V236I, V236N, V236L, V236M, and V236F) residue. The crystal structures of the mutants were determined, and the effects of the mutations on the rates of CO, O2, and NO binding were investigated. O2 reduction and NO binding were unaffected in V236T, while the oxidation of heme b during O−O bond cleavage was not detected in V236A. The V236A results are attributed to a decrease in the rate of electron transfer between heme b and heme a3 during O−O bond cleavage in V236A, followed by faster re-reduction of heme b by CuA. This interpretation is supported by classical molecular dynamics simulations of diffusion of O2 to the active site in V236A that indicated a larger distance between the two hemes compared to that in the wild type and increased contact of heme a3 with water and weakened interactions with residues R444 and R445. As the size of the mutant side chain increased and protruded more into the ligand cavity, the rates of ligand binding decreased correspondingly. These results demonstrate the importance of V236 in facilitating access of ligands to the active site in T. thermophilus ba3.

Spectral Identification of Intermediates Generated during the Reaction of Dioxygen with the Wild-type and EQ(I-286) Mutant of Rhodobacter Sphaeroides Cytochrome c Oxidase

Biochemistry 51(46) 9302–9311

Conserved Glycine 232 in the Ligand Channel of ba3 Cytochrome Oxidase from Thermus thermophilus

Biochemistry 53(27), 4467—4475

Knowing how the protein environment modulates ligand pathways and redox centers in the respiratory heme-copper oxidases is fundamental for understanding the relationship between the structure and function of these enzymes. In this study, we investigated the reactions of O2 and NO with the fully reduced G232V mutant of ba3 cytochrome c oxidase from Thermus thermophilus (Tt ba3) in which a conserved glycine residue in the O2 channel of the enzyme was replaced with a bulkier valine residue. Previous studies of the homologous mutant of Rhodobacter sphaeroides aa3 cytochrome c oxidase suggested that the valine completely blocked the access of O2 to the active site [Salomonsson, L., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 11617−11621]. Using photolabile O2 and NO carriers, we find by using time-resolved optical absorption spectroscopy that the rates of O2 and NO binding are not significantly affected in the Tt ba3 G232V mutant. Classical molecular dynamics simulations of diffusion of O2 to the active site in the wild-type enzyme and G232V mutant show that the insertion of the larger valine residue in place of the glycine appears to open up other O2 and NO exit/entrance pathways that allow these ligands unhindered access to the active site, thus compensating for the larger valine residue.

Ligand Access to the Active Site in Thermus thermophilus ba3 and Bovine Heart aa3 Cytochrome Oxidases.

Biochemistry 52, 640–652

Knowledge of the structure and dynamics of the ligand channel(s) in heme-copper oxidases is critical for understanding how the protein environment modulates the functions of these enzymes. Using photolabile NO and O2 carriers, we recently found that NO and O2 binding in Thermus thermophilus (Tt ) ba3 is ∼10 times faster than in the bovine enzyme, indicating that inherent structural differences affect ligand access in these enzymes. Using X-ray crystallog- raphy, time-resolved optical absorption measurements, and theoretical calculations, we investigated ligand access in wild- type Tt ba3 and the mutants, Y133W, T231F, and Y133W/T231F, in which tyrosine and threonine in the O2 channel of Tt ba3 are replaced by the corresponding bulkier tryptophan and phenylalanine, respectively, present in the aa3 enzymes. NO binding in Y133W and Y133W/T231F was found to be 5 times slower than in wild-type ba3 and the T231F mutant. The results show that the Tt ba3 Y133W mutation and the bovine W126 residue physically impede NO access to the binuclear center. In the bovine enzyme, there is a hydrophobic “way station”, which may further slow ligand access to the active site. Classical simulations of diffusion of Xe to the active sites in ba3 and bovine aa3 show conformational freedom of the bovine F238 and the F231 side chain of the Tt ba3 Y133W/T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme.

The CO Photodissociation and Recombination Dynamics of the W172Y/ F282T Ligand Channel Mutant of Rhodobacter sphaeroides aa3 Cytochrome c Oxidase

Photochemistry and Photobiology

In the ligand channel of the cytochrome c oxidase from Rhodobacter sphaeroides (Rs aa3) W172 and F282 have been proposed to generate a constriction that may slow ligand access to and from the active site. To explore this issue, the tryptophan and phenylalanine residues in Rs aa3 were mutated to the less bulky tyrosine and threonine residues, respectively, which occupy these sites in Thermus thermophilus (Tt) ba3 cytochrome oxidase. The CO photolysis and recombination dynamics of the reduced wild-type Rs aa3 and the W172Y/F282T mutant were investigated using time-resolved optical absorption spectroscopy. The spectral changes associated with the multiple processes are attributed to different conformers. The major CO recombination process (44 μs) in the W172Y/F282T mutant is ~500 times faster than the predominant CO recombination process in the wild-type enzyme (~23 ms). Classical dynamic simulations of the wild-type enzyme and double mutant showed significant structural changes at the active site in the mutant, including movement of the heme a3 ring-D propionate toward CuB and reduced binuclear center cavity volume. These structural changes effectively close the ligand exit pathway from the binuclear center, providing a basis for the faster CO recombination in the double mutant.

Kinetics and Intermediates of the Reaction of Fully Reduced Escherichia coli bo3 Ubiquinol Oxidase with O2

Biochemistry 53(33), 5393—5404

Cytochrome bo3 ubiquinol oxidase from Escherichia coli catalyzes the reduction of O2 to water by ubiquinol. The reaction mechanism and the role of ubiquinol continue to be a subject of discussion. In this study, we report a detailed kinetic scheme of the reaction of cytochrome bo3 with O2 with steps specific to ubiquinol. The reaction was investigated using the CO flow-flash method, and time-resolved optical absorption difference spectra were collected from 1 μs to 20 ms after photolysis. Singular value decomposition-based global exponential fitting resolved five apparent lifetimes, 22 μs, 30 μs, 42 μs, 470 μs, and 2.0 ms. The reaction mechanism was derived by an algebraic kinetic analysis method using frequency-shifted spectra of known bovine states to identify the bo3 intermediates. It shows 42 μs O2 binding (3.8 × 107 M−1 s−1), producing compound A, followed by faster (22 μs) heme b oxidation, yielding a mixture of PR and F, and rapid heme b rereduction by ubiquinol (30 μs), producing the F intermediate and semiquinone. In the 470 μs step, the o3 F state is converted into the o33+ oxidized state, presumably by semiquinone/ubiquinol, without the concomitant oxidation of heme b. The final 2 ms step shows heme b reoxidation and the partial rereduction of the binuclear center and, following O2 binding, the formation of a mixture of P and F during a second turnover cycle. The results show that ubiquinol/semiquinone plays a complex role in the mechanism of O2 reduction by bo3, displaying kinetic steps that have no analogy in the CuA-containing heme-copper oxidases.

Role of the Conserved Valine 236 in Access of Ligands to the Active Site of Thermus thermophilus ba3 Cytochrome Oxidase

Biochemistry

Knowledge of the role of conserved residues in the ligand channel of heme-copper oxidases is critical for understanding how the protein scaffold modulates the function of these enzymes. In this study, we investigated the role of the conserved valine 236 in the ligand channel of ba3 cytochrome c oxidase from Thermus thermophilus by mutating the residue to a more polar (V236T), smaller (V236A), or larger (V236I, V236N, V236L, V236M, and V236F) residue. The crystal structures of the mutants were determined, and the effects of the mutations on the rates of CO, O2, and NO binding were investigated. O2 reduction and NO binding were unaffected in V236T, while the oxidation of heme b during O−O bond cleavage was not detected in V236A. The V236A results are attributed to a decrease in the rate of electron transfer between heme b and heme a3 during O−O bond cleavage in V236A, followed by faster re-reduction of heme b by CuA. This interpretation is supported by classical molecular dynamics simulations of diffusion of O2 to the active site in V236A that indicated a larger distance between the two hemes compared to that in the wild type and increased contact of heme a3 with water and weakened interactions with residues R444 and R445. As the size of the mutant side chain increased and protruded more into the ligand cavity, the rates of ligand binding decreased correspondingly. These results demonstrate the importance of V236 in facilitating access of ligands to the active site in T. thermophilus ba3.

The pathway of O2 to the active site in heme–copper oxidases

Biochimica et Biophysica Acta—Bioenergetics 1847(1), 109—118

The route of O2 to and from the high-spin heme in heme–copper oxidases has generally been believed to emulate that of carbon monoxide (CO). Time-resolved and stationary infrared experiments in our laboratories of the fully reduced CO-bound enzymes, as well as transient optical absorption saturation kinetics studies as a function of CO pressure, have provided strong support for CO binding to CuB on the pathway to and from the high-spin heme. The presence of CO on CuB suggests that O2 binding may be compromised in CO flow-flash experiments. Time- resolved optical absorption studies show that the rate of O2 and NO binding in the bovine enzyme is unaffected by the presence of CO, which is consistent with the rapid dissociation of CO from CuB . In contrast, in Thermus thermophilus (Tt) cytochrome ba3 the O2 and NO binding to heme a3 slows by an order of magnitude in the presence of CO, but is still considerably faster than the CO off-rate from CuB in the absence of O2. These results show that traditional CO flow-flash experiments do not give accurate results for the physiological binding of O2 and NO in Tt ba3, namely, in the absence of CO. They also raise the question whether in CO flow-flash experiments on Tt ba3 the presence of CO on CuB impedes the binding of O2 to CuB or, if O2 does not bind to CuB prior to heme a3, whether the CuB –CO complex sterically restricts access of O2 to the heme. Both possibilities are discussed, and we argue that O2 binds directly to heme a3 in Tt ba3, causing CO to dissociate from CuB in a concerted manner through steric and/or electronic effects. This would allow CuB to function as an electron donor during the fast breaking of the O-O bond. These results suggest that the binding of CO to CuB on the path to and from heme a3 may not be applicable to O2 and NO in all heme-copper oxidases.

The pathway of O2 to the active site in heme–copper oxidases

Biochimica et Biophysica Acta

The route of O2 to and from the high-spin heme in heme–copper oxidases has generally been believed to emulate that of carbon monoxide (CO). Time-resolved and stationary infrared experiments in our laboratories of the fully reduced CO-bound enzymes, as well as transient optical absorption saturation kinetics studies as a function of CO pressure, have provided strong support for CO binding to Cu+B on the pathway to and from the high-spin heme. The presence of CO on Cu+B suggests that O2 binding may be compromised in CO flow-flash experiments. Time- resolved optical absorption studies show that the rate of O2 and NO binding in the bovine enzyme (1 × 108 M− 1 s− 1) is unaffected by the presence of CO, which is consistent with the rapid dissociation (t1/2 = 1.5 μs) of CO from Cu+B . In contrast, in Thermus thermophilus (Tt) cytochrome ba3 the O2 and NO binding to heme a3 slows by an order of magnitude in the presence of CO (from 1 × 109 to 1 × 108 M−1 s−1), but is still considerably faster (~10 μs at 1 atm O2) than the CO off-rate from CuB in the absence of O2 (milliseconds). These results show that traditional CO flow-flash experiments do not give accurate results for the physiological binding of O2 and NO in Tt ba3, namely, in the absence of CO. They also raise the question whether in CO flow-flash experiments on Tt ba3 the presence of CO on Cu+B impedes the binding of O2 to Cu+B or, if O2 does not bind to Cu+B prior to heme a3, whether the Cu+B –CO complex sterically restricts access of O2 to the heme. Both possibilities are discussed, and we argue that O2 binds directly to heme a3 in Tt ba3, causing CO to dissociate from Cu+B in a concerted manner through steric and/or electronic effects. This would allow Cu+B to function as an electron donor during the fast (5 μs) breaking of the O\O bond. These results suggest that the binding of CO to Cu+B on the path to and from heme a3 may not be applicable to O2 and NO in all heme-copper oxidases.

Spectral Identification of Intermediates Generated during the Reaction of Dioxygen with the Wild-type and EQ(I-286) Mutant of Rhodobacter Sphaeroides Cytochrome c Oxidase

Biochemistry 51(46) 9302–9311

Conserved Glycine 232 in the Ligand Channel of ba3 Cytochrome Oxidase from Thermus thermophilus

Biochemistry 53(27), 4467—4475

Knowing how the protein environment modulates ligand pathways and redox centers in the respiratory heme-copper oxidases is fundamental for understanding the relationship between the structure and function of these enzymes. In this study, we investigated the reactions of O2 and NO with the fully reduced G232V mutant of ba3 cytochrome c oxidase from Thermus thermophilus (Tt ba3) in which a conserved glycine residue in the O2 channel of the enzyme was replaced with a bulkier valine residue. Previous studies of the homologous mutant of Rhodobacter sphaeroides aa3 cytochrome c oxidase suggested that the valine completely blocked the access of O2 to the active site [Salomonsson, L., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 11617−11621]. Using photolabile O2 and NO carriers, we find by using time-resolved optical absorption spectroscopy that the rates of O2 and NO binding are not significantly affected in the Tt ba3 G232V mutant. Classical molecular dynamics simulations of diffusion of O2 to the active site in the wild-type enzyme and G232V mutant show that the insertion of the larger valine residue in place of the glycine appears to open up other O2 and NO exit/entrance pathways that allow these ligands unhindered access to the active site, thus compensating for the larger valine residue.

Ligand Access to the Active Site in Thermus thermophilus ba3 and Bovine Heart aa3 Cytochrome Oxidases.

Biochemistry 52, 640–652

Knowledge of the structure and dynamics of the ligand channel(s) in heme-copper oxidases is critical for understanding how the protein environment modulates the functions of these enzymes. Using photolabile NO and O2 carriers, we recently found that NO and O2 binding in Thermus thermophilus (Tt ) ba3 is ∼10 times faster than in the bovine enzyme, indicating that inherent structural differences affect ligand access in these enzymes. Using X-ray crystallog- raphy, time-resolved optical absorption measurements, and theoretical calculations, we investigated ligand access in wild- type Tt ba3 and the mutants, Y133W, T231F, and Y133W/T231F, in which tyrosine and threonine in the O2 channel of Tt ba3 are replaced by the corresponding bulkier tryptophan and phenylalanine, respectively, present in the aa3 enzymes. NO binding in Y133W and Y133W/T231F was found to be 5 times slower than in wild-type ba3 and the T231F mutant. The results show that the Tt ba3 Y133W mutation and the bovine W126 residue physically impede NO access to the binuclear center. In the bovine enzyme, there is a hydrophobic “way station”, which may further slow ligand access to the active site. Classical simulations of diffusion of Xe to the active sites in ba3 and bovine aa3 show conformational freedom of the bovine F238 and the F231 side chain of the Tt ba3 Y133W/T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme.

The CO Photodissociation and Recombination Dynamics of the W172Y/ F282T Ligand Channel Mutant of Rhodobacter sphaeroides aa3 Cytochrome c Oxidase

Photochemistry and Photobiology

In the ligand channel of the cytochrome c oxidase from Rhodobacter sphaeroides (Rs aa3) W172 and F282 have been proposed to generate a constriction that may slow ligand access to and from the active site. To explore this issue, the tryptophan and phenylalanine residues in Rs aa3 were mutated to the less bulky tyrosine and threonine residues, respectively, which occupy these sites in Thermus thermophilus (Tt) ba3 cytochrome oxidase. The CO photolysis and recombination dynamics of the reduced wild-type Rs aa3 and the W172Y/F282T mutant were investigated using time-resolved optical absorption spectroscopy. The spectral changes associated with the multiple processes are attributed to different conformers. The major CO recombination process (44 μs) in the W172Y/F282T mutant is ~500 times faster than the predominant CO recombination process in the wild-type enzyme (~23 ms). Classical dynamic simulations of the wild-type enzyme and double mutant showed significant structural changes at the active site in the mutant, including movement of the heme a3 ring-D propionate toward CuB and reduced binuclear center cavity volume. These structural changes effectively close the ligand exit pathway from the binuclear center, providing a basis for the faster CO recombination in the double mutant.

Kinetics and Intermediates of the Reaction of Fully Reduced Escherichia coli bo3 Ubiquinol Oxidase with O2

Biochemistry 53(33), 5393—5404

Cytochrome bo3 ubiquinol oxidase from Escherichia coli catalyzes the reduction of O2 to water by ubiquinol. The reaction mechanism and the role of ubiquinol continue to be a subject of discussion. In this study, we report a detailed kinetic scheme of the reaction of cytochrome bo3 with O2 with steps specific to ubiquinol. The reaction was investigated using the CO flow-flash method, and time-resolved optical absorption difference spectra were collected from 1 μs to 20 ms after photolysis. Singular value decomposition-based global exponential fitting resolved five apparent lifetimes, 22 μs, 30 μs, 42 μs, 470 μs, and 2.0 ms. The reaction mechanism was derived by an algebraic kinetic analysis method using frequency-shifted spectra of known bovine states to identify the bo3 intermediates. It shows 42 μs O2 binding (3.8 × 107 M−1 s−1), producing compound A, followed by faster (22 μs) heme b oxidation, yielding a mixture of PR and F, and rapid heme b rereduction by ubiquinol (30 μs), producing the F intermediate and semiquinone. In the 470 μs step, the o3 F state is converted into the o33+ oxidized state, presumably by semiquinone/ubiquinol, without the concomitant oxidation of heme b. The final 2 ms step shows heme b reoxidation and the partial rereduction of the binuclear center and, following O2 binding, the formation of a mixture of P and F during a second turnover cycle. The results show that ubiquinol/semiquinone plays a complex role in the mechanism of O2 reduction by bo3, displaying kinetic steps that have no analogy in the CuA-containing heme-copper oxidases.

Role of the Conserved Valine 236 in Access of Ligands to the Active Site of Thermus thermophilus ba3 Cytochrome Oxidase

Biochemistry

Knowledge of the role of conserved residues in the ligand channel of heme-copper oxidases is critical for understanding how the protein scaffold modulates the function of these enzymes. In this study, we investigated the role of the conserved valine 236 in the ligand channel of ba3 cytochrome c oxidase from Thermus thermophilus by mutating the residue to a more polar (V236T), smaller (V236A), or larger (V236I, V236N, V236L, V236M, and V236F) residue. The crystal structures of the mutants were determined, and the effects of the mutations on the rates of CO, O2, and NO binding were investigated. O2 reduction and NO binding were unaffected in V236T, while the oxidation of heme b during O−O bond cleavage was not detected in V236A. The V236A results are attributed to a decrease in the rate of electron transfer between heme b and heme a3 during O−O bond cleavage in V236A, followed by faster re-reduction of heme b by CuA. This interpretation is supported by classical molecular dynamics simulations of diffusion of O2 to the active site in V236A that indicated a larger distance between the two hemes compared to that in the wild type and increased contact of heme a3 with water and weakened interactions with residues R444 and R445. As the size of the mutant side chain increased and protruded more into the ligand cavity, the rates of ligand binding decreased correspondingly. These results demonstrate the importance of V236 in facilitating access of ligands to the active site in T. thermophilus ba3.

The pathway of O2 to the active site in heme–copper oxidases

Biochimica et Biophysica Acta—Bioenergetics 1847(1), 109—118

The route of O2 to and from the high-spin heme in heme–copper oxidases has generally been believed to emulate that of carbon monoxide (CO). Time-resolved and stationary infrared experiments in our laboratories of the fully reduced CO-bound enzymes, as well as transient optical absorption saturation kinetics studies as a function of CO pressure, have provided strong support for CO binding to CuB on the pathway to and from the high-spin heme. The presence of CO on CuB suggests that O2 binding may be compromised in CO flow-flash experiments. Time- resolved optical absorption studies show that the rate of O2 and NO binding in the bovine enzyme is unaffected by the presence of CO, which is consistent with the rapid dissociation of CO from CuB . In contrast, in Thermus thermophilus (Tt) cytochrome ba3 the O2 and NO binding to heme a3 slows by an order of magnitude in the presence of CO, but is still considerably faster than the CO off-rate from CuB in the absence of O2. These results show that traditional CO flow-flash experiments do not give accurate results for the physiological binding of O2 and NO in Tt ba3, namely, in the absence of CO. They also raise the question whether in CO flow-flash experiments on Tt ba3 the presence of CO on CuB impedes the binding of O2 to CuB or, if O2 does not bind to CuB prior to heme a3, whether the CuB –CO complex sterically restricts access of O2 to the heme. Both possibilities are discussed, and we argue that O2 binds directly to heme a3 in Tt ba3, causing CO to dissociate from CuB in a concerted manner through steric and/or electronic effects. This would allow CuB to function as an electron donor during the fast breaking of the O-O bond. These results suggest that the binding of CO to CuB on the path to and from heme a3 may not be applicable to O2 and NO in all heme-copper oxidases.

The pathway of O2 to the active site in heme–copper oxidases

Biochimica et Biophysica Acta

The route of O2 to and from the high-spin heme in heme–copper oxidases has generally been believed to emulate that of carbon monoxide (CO). Time-resolved and stationary infrared experiments in our laboratories of the fully reduced CO-bound enzymes, as well as transient optical absorption saturation kinetics studies as a function of CO pressure, have provided strong support for CO binding to Cu+B on the pathway to and from the high-spin heme. The presence of CO on Cu+B suggests that O2 binding may be compromised in CO flow-flash experiments. Time- resolved optical absorption studies show that the rate of O2 and NO binding in the bovine enzyme (1 × 108 M− 1 s− 1) is unaffected by the presence of CO, which is consistent with the rapid dissociation (t1/2 = 1.5 μs) of CO from Cu+B . In contrast, in Thermus thermophilus (Tt) cytochrome ba3 the O2 and NO binding to heme a3 slows by an order of magnitude in the presence of CO (from 1 × 109 to 1 × 108 M−1 s−1), but is still considerably faster (~10 μs at 1 atm O2) than the CO off-rate from CuB in the absence of O2 (milliseconds). These results show that traditional CO flow-flash experiments do not give accurate results for the physiological binding of O2 and NO in Tt ba3, namely, in the absence of CO. They also raise the question whether in CO flow-flash experiments on Tt ba3 the presence of CO on Cu+B impedes the binding of O2 to Cu+B or, if O2 does not bind to Cu+B prior to heme a3, whether the Cu+B –CO complex sterically restricts access of O2 to the heme. Both possibilities are discussed, and we argue that O2 binds directly to heme a3 in Tt ba3, causing CO to dissociate from Cu+B in a concerted manner through steric and/or electronic effects. This would allow Cu+B to function as an electron donor during the fast (5 μs) breaking of the O\O bond. These results suggest that the binding of CO to Cu+B on the path to and from heme a3 may not be applicable to O2 and NO in all heme-copper oxidases.

THEORETICAL STUDIES OF THE FUNCTIONAL ROLE OF THE CROSS-LINKED HISTIDINE-TYROSINE COPPER B LIGAND OF CYTOCHROME C OXIDASE

ProQuest Dissertations & Theses

This work predicts the spectroscopic and thermodynamic properties of chemical models of the histidine-tyrosine CuB ligand at the active site of cytochrome c oxidase using quantum chemical calculations (DFT and CASSCF). Vibrational and UV/Vis spectra are predicted, and the change in pKa, redox potential, and bond dissociation energy of the phenolic-OH compared to the unmodified phenol are calculated, both with and without copper(II) ion.

Spectral Identification of Intermediates Generated during the Reaction of Dioxygen with the Wild-type and EQ(I-286) Mutant of Rhodobacter Sphaeroides Cytochrome c Oxidase

Biochemistry 51(46) 9302–9311

Conserved Glycine 232 in the Ligand Channel of ba3 Cytochrome Oxidase from Thermus thermophilus

Biochemistry 53(27), 4467—4475

Knowing how the protein environment modulates ligand pathways and redox centers in the respiratory heme-copper oxidases is fundamental for understanding the relationship between the structure and function of these enzymes. In this study, we investigated the reactions of O2 and NO with the fully reduced G232V mutant of ba3 cytochrome c oxidase from Thermus thermophilus (Tt ba3) in which a conserved glycine residue in the O2 channel of the enzyme was replaced with a bulkier valine residue. Previous studies of the homologous mutant of Rhodobacter sphaeroides aa3 cytochrome c oxidase suggested that the valine completely blocked the access of O2 to the active site [Salomonsson, L., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 11617−11621]. Using photolabile O2 and NO carriers, we find by using time-resolved optical absorption spectroscopy that the rates of O2 and NO binding are not significantly affected in the Tt ba3 G232V mutant. Classical molecular dynamics simulations of diffusion of O2 to the active site in the wild-type enzyme and G232V mutant show that the insertion of the larger valine residue in place of the glycine appears to open up other O2 and NO exit/entrance pathways that allow these ligands unhindered access to the active site, thus compensating for the larger valine residue.

Ligand Access to the Active Site in Thermus thermophilus ba3 and Bovine Heart aa3 Cytochrome Oxidases.

Biochemistry 52, 640–652

Knowledge of the structure and dynamics of the ligand channel(s) in heme-copper oxidases is critical for understanding how the protein environment modulates the functions of these enzymes. Using photolabile NO and O2 carriers, we recently found that NO and O2 binding in Thermus thermophilus (Tt ) ba3 is ∼10 times faster than in the bovine enzyme, indicating that inherent structural differences affect ligand access in these enzymes. Using X-ray crystallog- raphy, time-resolved optical absorption measurements, and theoretical calculations, we investigated ligand access in wild- type Tt ba3 and the mutants, Y133W, T231F, and Y133W/T231F, in which tyrosine and threonine in the O2 channel of Tt ba3 are replaced by the corresponding bulkier tryptophan and phenylalanine, respectively, present in the aa3 enzymes. NO binding in Y133W and Y133W/T231F was found to be 5 times slower than in wild-type ba3 and the T231F mutant. The results show that the Tt ba3 Y133W mutation and the bovine W126 residue physically impede NO access to the binuclear center. In the bovine enzyme, there is a hydrophobic “way station”, which may further slow ligand access to the active site. Classical simulations of diffusion of Xe to the active sites in ba3 and bovine aa3 show conformational freedom of the bovine F238 and the F231 side chain of the Tt ba3 Y133W/T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme.

The CO Photodissociation and Recombination Dynamics of the W172Y/ F282T Ligand Channel Mutant of Rhodobacter sphaeroides aa3 Cytochrome c Oxidase

Photochemistry and Photobiology

In the ligand channel of the cytochrome c oxidase from Rhodobacter sphaeroides (Rs aa3) W172 and F282 have been proposed to generate a constriction that may slow ligand access to and from the active site. To explore this issue, the tryptophan and phenylalanine residues in Rs aa3 were mutated to the less bulky tyrosine and threonine residues, respectively, which occupy these sites in Thermus thermophilus (Tt) ba3 cytochrome oxidase. The CO photolysis and recombination dynamics of the reduced wild-type Rs aa3 and the W172Y/F282T mutant were investigated using time-resolved optical absorption spectroscopy. The spectral changes associated with the multiple processes are attributed to different conformers. The major CO recombination process (44 μs) in the W172Y/F282T mutant is ~500 times faster than the predominant CO recombination process in the wild-type enzyme (~23 ms). Classical dynamic simulations of the wild-type enzyme and double mutant showed significant structural changes at the active site in the mutant, including movement of the heme a3 ring-D propionate toward CuB and reduced binuclear center cavity volume. These structural changes effectively close the ligand exit pathway from the binuclear center, providing a basis for the faster CO recombination in the double mutant.

Kinetics and Intermediates of the Reaction of Fully Reduced Escherichia coli bo3 Ubiquinol Oxidase with O2

Biochemistry 53(33), 5393—5404

Cytochrome bo3 ubiquinol oxidase from Escherichia coli catalyzes the reduction of O2 to water by ubiquinol. The reaction mechanism and the role of ubiquinol continue to be a subject of discussion. In this study, we report a detailed kinetic scheme of the reaction of cytochrome bo3 with O2 with steps specific to ubiquinol. The reaction was investigated using the CO flow-flash method, and time-resolved optical absorption difference spectra were collected from 1 μs to 20 ms after photolysis. Singular value decomposition-based global exponential fitting resolved five apparent lifetimes, 22 μs, 30 μs, 42 μs, 470 μs, and 2.0 ms. The reaction mechanism was derived by an algebraic kinetic analysis method using frequency-shifted spectra of known bovine states to identify the bo3 intermediates. It shows 42 μs O2 binding (3.8 × 107 M−1 s−1), producing compound A, followed by faster (22 μs) heme b oxidation, yielding a mixture of PR and F, and rapid heme b rereduction by ubiquinol (30 μs), producing the F intermediate and semiquinone. In the 470 μs step, the o3 F state is converted into the o33+ oxidized state, presumably by semiquinone/ubiquinol, without the concomitant oxidation of heme b. The final 2 ms step shows heme b reoxidation and the partial rereduction of the binuclear center and, following O2 binding, the formation of a mixture of P and F during a second turnover cycle. The results show that ubiquinol/semiquinone plays a complex role in the mechanism of O2 reduction by bo3, displaying kinetic steps that have no analogy in the CuA-containing heme-copper oxidases.

Role of the Conserved Valine 236 in Access of Ligands to the Active Site of Thermus thermophilus ba3 Cytochrome Oxidase

Biochemistry

Knowledge of the role of conserved residues in the ligand channel of heme-copper oxidases is critical for understanding how the protein scaffold modulates the function of these enzymes. In this study, we investigated the role of the conserved valine 236 in the ligand channel of ba3 cytochrome c oxidase from Thermus thermophilus by mutating the residue to a more polar (V236T), smaller (V236A), or larger (V236I, V236N, V236L, V236M, and V236F) residue. The crystal structures of the mutants were determined, and the effects of the mutations on the rates of CO, O2, and NO binding were investigated. O2 reduction and NO binding were unaffected in V236T, while the oxidation of heme b during O−O bond cleavage was not detected in V236A. The V236A results are attributed to a decrease in the rate of electron transfer between heme b and heme a3 during O−O bond cleavage in V236A, followed by faster re-reduction of heme b by CuA. This interpretation is supported by classical molecular dynamics simulations of diffusion of O2 to the active site in V236A that indicated a larger distance between the two hemes compared to that in the wild type and increased contact of heme a3 with water and weakened interactions with residues R444 and R445. As the size of the mutant side chain increased and protruded more into the ligand cavity, the rates of ligand binding decreased correspondingly. These results demonstrate the importance of V236 in facilitating access of ligands to the active site in T. thermophilus ba3.

The pathway of O2 to the active site in heme–copper oxidases

Biochimica et Biophysica Acta—Bioenergetics 1847(1), 109—118

The route of O2 to and from the high-spin heme in heme–copper oxidases has generally been believed to emulate that of carbon monoxide (CO). Time-resolved and stationary infrared experiments in our laboratories of the fully reduced CO-bound enzymes, as well as transient optical absorption saturation kinetics studies as a function of CO pressure, have provided strong support for CO binding to CuB on the pathway to and from the high-spin heme. The presence of CO on CuB suggests that O2 binding may be compromised in CO flow-flash experiments. Time- resolved optical absorption studies show that the rate of O2 and NO binding in the bovine enzyme is unaffected by the presence of CO, which is consistent with the rapid dissociation of CO from CuB . In contrast, in Thermus thermophilus (Tt) cytochrome ba3 the O2 and NO binding to heme a3 slows by an order of magnitude in the presence of CO, but is still considerably faster than the CO off-rate from CuB in the absence of O2. These results show that traditional CO flow-flash experiments do not give accurate results for the physiological binding of O2 and NO in Tt ba3, namely, in the absence of CO. They also raise the question whether in CO flow-flash experiments on Tt ba3 the presence of CO on CuB impedes the binding of O2 to CuB or, if O2 does not bind to CuB prior to heme a3, whether the CuB –CO complex sterically restricts access of O2 to the heme. Both possibilities are discussed, and we argue that O2 binds directly to heme a3 in Tt ba3, causing CO to dissociate from CuB in a concerted manner through steric and/or electronic effects. This would allow CuB to function as an electron donor during the fast breaking of the O-O bond. These results suggest that the binding of CO to CuB on the path to and from heme a3 may not be applicable to O2 and NO in all heme-copper oxidases.

The pathway of O2 to the active site in heme–copper oxidases

Biochimica et Biophysica Acta

The route of O2 to and from the high-spin heme in heme–copper oxidases has generally been believed to emulate that of carbon monoxide (CO). Time-resolved and stationary infrared experiments in our laboratories of the fully reduced CO-bound enzymes, as well as transient optical absorption saturation kinetics studies as a function of CO pressure, have provided strong support for CO binding to Cu+B on the pathway to and from the high-spin heme. The presence of CO on Cu+B suggests that O2 binding may be compromised in CO flow-flash experiments. Time- resolved optical absorption studies show that the rate of O2 and NO binding in the bovine enzyme (1 × 108 M− 1 s− 1) is unaffected by the presence of CO, which is consistent with the rapid dissociation (t1/2 = 1.5 μs) of CO from Cu+B . In contrast, in Thermus thermophilus (Tt) cytochrome ba3 the O2 and NO binding to heme a3 slows by an order of magnitude in the presence of CO (from 1 × 109 to 1 × 108 M−1 s−1), but is still considerably faster (~10 μs at 1 atm O2) than the CO off-rate from CuB in the absence of O2 (milliseconds). These results show that traditional CO flow-flash experiments do not give accurate results for the physiological binding of O2 and NO in Tt ba3, namely, in the absence of CO. They also raise the question whether in CO flow-flash experiments on Tt ba3 the presence of CO on Cu+B impedes the binding of O2 to Cu+B or, if O2 does not bind to Cu+B prior to heme a3, whether the Cu+B –CO complex sterically restricts access of O2 to the heme. Both possibilities are discussed, and we argue that O2 binds directly to heme a3 in Tt ba3, causing CO to dissociate from Cu+B in a concerted manner through steric and/or electronic effects. This would allow Cu+B to function as an electron donor during the fast (5 μs) breaking of the O\O bond. These results suggest that the binding of CO to Cu+B on the path to and from heme a3 may not be applicable to O2 and NO in all heme-copper oxidases.

THEORETICAL STUDIES OF THE FUNCTIONAL ROLE OF THE CROSS-LINKED HISTIDINE-TYROSINE COPPER B LIGAND OF CYTOCHROME C OXIDASE

ProQuest Dissertations & Theses

This work predicts the spectroscopic and thermodynamic properties of chemical models of the histidine-tyrosine CuB ligand at the active site of cytochrome c oxidase using quantum chemical calculations (DFT and CASSCF). Vibrational and UV/Vis spectra are predicted, and the change in pKa, redox potential, and bond dissociation energy of the phenolic-OH compared to the unmodified phenol are calculated, both with and without copper(II) ion.

Mild and Expedient Asymmetric Reductions of α,β-Unsaturated Alkenyl and Alkynyl Ketons by TarB-NO2 and Mechanistic Investigations of Ketone Reduction

Journal of Organic Chemistry 75, 7717–7725

A facile and mild reduction procedure is reported for the preparation of chiral allylic and propargyl alcohols in high enantiomeric purity. Under optimized conditions, alkynyl and alkenyl ketones were reduced by TarB-NO(2) and NaBH(4) at 25 °C in 1 h to produce chiral propargyl and allylic alcohols with enantiomeric excesses and yields up to 99%. In the case of α,β-unsaturated alkenyl ketones, α-substituted cycloalkenones were reduced with up to 99% ee, while more substituted and acyclic derivatives exhibited lower induction. For α,β-ynones, it was found that highly branched aliphatic ynones were reduced with optimal induction up to 90% ee, while reduction of aromatic and linear aliphatic derivatives resulted in more modest enantioselectivity. Using the (l)-TarB-NO(2) reagent derived from (l)-tartaric acid, we routinely obtained highly enantioenriched chiral allylic and propargyl alcohols with (R) configuration. Since previous models and a reduction of a saturated analogue predicted propargyl products of (S) configuration, a series of new mechanistic studies were conducted to determine the likely orientation of aromatic, alkenyl, and alkynyl ketones in the transition state.