title: The cytochrome ba3 oxygen reductase from Thermus thermophilus uses a single input channel for proton delivery to the active site and for proton pumping.
abstract: The heme-copper oxygen reductases are redox-driven proton pumps that generate a proton motive force in both prokaryotes and mitochondria. These enzymes have been divided into 3 evolutionarily related groups: the A-, B- and C-families. Most experimental work on proton-pumping mechanisms has been performed with members of the A-family. These enzymes require 2 proton input pathways (D- and K-channels) to transfer protons used for oxygen reduction chemistry and for proton pumping, with the D-channel transporting all pumped protons. In this work we use site-directed mutagenesis to demonstrate that the ba(3) oxygen reductase from Thermus thermophilus, a representative of the B-family, does not contain a D-channel. Rather, it utilizes only 1 proton input channel, analogous to that of the A-family K-channel, and it delivers protons to the active site for both O2 chemistry and proton pumping. Comparison of available subunit I sequences reveals that the only structural elements conserved within the oxygen reductase families that could perform these functions are active-site components, namely the covalently linked histidine-tyrosine, the Cu(B) and its ligands, and the active-site heme and its ligands. Therefore, our data suggest that all oxygen reductases perform the same chemical reactions for oxygen reduction and comprise the essential elements of the proton-pumping mechanism (e.g., the proton-loading and kinetic-gating sites). These sites, however, cannot be located within the D-channel. These results along with structural considerations point to the A-propionate region of the active-site heme and surrounding water molecules as the proton-loading site.
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Hsin-Yang
authors.initials: HY
authors.lastname: Chang
authors.forename: James
authors.initials: J
authors.lastname: Hemp
authors.forename: Ying
authors.initials: Y
authors.lastname: Chen
authors.forename: James A
authors.initials: JA
authors.lastname: Fee
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003579:Cytochrome Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003573:Cytochrome b Group / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017494:Proton Pumps / Q000235:genetics / Q000378:metabolism; D011522:Protons; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003573:Cytochrome b Group; D017494:Proton Pumps; D011522:Protons; C058557:cytochrome ba3; D003579:Cytochrome Reductases; D003576:Electron Transport Complex IV; D010100:Oxygen