title: Plasticity in the High Affinity Menaquinone Binding Site of the Cytochrome aa3-600 Menaquinol Oxidase from Bacillus subtilis.
abstract: Cytochrome aa3-600 is a terminal oxidase in the electron transport pathway that contributes to the electrochemical membrane potential by actively pumping protons. A notable feature of this enzyme complex is that it uses menaquinol as its electron donor instead of cytochrome c when it reduces dioxygen to water. The enzyme stabilizes a menasemiquinone radical (SQ) at a high affinity site that is important for catalysis. One of the residues that interacts with the semiquinone is Arg70. We have made the R70H mutant and have characterized the menasemiquinone radical by advanced X- and Q-band EPR. The bound SQ of the R70H mutant exhibits a strong isotropic hyperfine coupling (a(14)N ≈ 2.0 MHz) with a hydrogen bonded nitrogen. This nitrogen originates from a histidine side chain, based on its quadrupole coupling constant, e(2)qQ/h = 1.44 MHz, typical for protonated imidazole nitrogens. In the wild-type cyt aa3-600, the SQ is instead hydrogen bonded with Nε from the Arg70 side chain. Analysis of the (1)H 2D electron spin echo envelope modulation (ESEEM) spectra shows that the mutation also changes the number and strength of the hydrogen bonds between the SQ and the surrounding protein. Despite the alterations in the immediate environment of the SQ, the R70H mutant remains catalytically active. These findings are in contrast to the equivalent mutation in the close homologue, cytochrome bo3 ubiquinol oxidase from Escherichia coli, where the R71H mutation eliminates function.
authors.affiliation: §Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
authors.forename: Sophia M
authors.initials: SM
authors.lastname: Yi
authors.affiliation: †Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.|‡Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
authors.forename: Alexander T
authors.initials: AT
authors.lastname: Taguchi
authors.affiliation: ⊥V. V. Voevodsky Institute of Chemical Kinetics and Combustion, Russian Academy of Sciences, Novosibirsk 630090, Russian Federation.
authors.forename: Rimma I
authors.initials: RI
authors.lastname: Samoilova
authors.affiliation: ∥School of Chemistry, The University of Manchester, Manchester M13 9PL, U.K.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: O'Malley
authors.affiliation: §Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.|†Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
authors.affiliation: ‡Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
authors.forename: Sergei A
authors.initials: SA
authors.lastname: Dikanov
mesh_terms: D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010971:Plastoquinone / Q000031:analogs & derivatives / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024482:Vitamin K 2 / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C083850:cytochrome bo3, E coli; C100461:menasemiquinone; D024482:Vitamin K 2; C082578:menaquinol oxidase; D003576:Electron Transport Complex IV; D010971:Plastoquinone