title: Role of the Conserved Valine 236 in Access of Ligands to the Active Site of Thermus thermophilus ba3Cytochrome Oxidase.
abstract: 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 ba3cytochrome 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. O2reduction 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 a3during 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 O2to 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 a3with 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California , Santa Cruz, California 95064, United States.
authors.forename: Chie
authors.identifier: http://orcid.org/0000-0002-9355-8155
authors.initials: C
authors.lastname: Funatogawa
authors.affiliation: Department of Molecular Biology, The Scripps Institute , MB-8, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Molecular Biology, The Scripps Institute , MB-8, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Ying
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Chemistry and Biochemistry, University of California , Santa Cruz, California 95064, United States.
authors.forename: William
authors.initials: W
authors.lastname: McDonald
authors.affiliation: Department of Chemistry and Biochemistry, University of California , Santa Cruz, California 95064, United States.
authors.forename: Istvan
authors.initials: I
authors.lastname: Szundi
authors.affiliation: Department of Molecular Biology, The Scripps Institute , MB-8, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: James A
authors.initials: JA
authors.lastname: Fee
authors.affiliation: Department of Molecular Biology, The Scripps Institute , MB-8, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: C David
authors.initials: CD
authors.lastname: Stout
authors.affiliation: Department of Chemistry and Biochemistry, University of California , Santa Cruz, California 95064, United States.
authors.forename: Ólöf
authors.identifier: http://orcid.org/0000-0002-9497-7079
authors.initials: Ó
authors.lastname: Einarsdóttir
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D003460:Crystallization; D018360:Crystallography, X-Ray; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D013053:Spectrophotometry; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics; D014633:Valine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003573:Cytochrome b Group; D008024:Ligands; D009569:Nitric Oxide; D006418:Heme; D002248:Carbon Monoxide; C058557:cytochrome ba3; D003576:Electron Transport Complex IV; D014633:Valine; D010100:Oxygen