title: The rate-limiting step in O(2) reduction by cytochrome ba(3) from Thermus thermophilus.
abstract: Cytochrome ba(3) (ba(3)) of Thermus thermophilus (T. thermophilus) is a member of the heme-copper oxidase family, which has a binuclear catalytic center comprised of a heme (heme a(3)) and a copper (Cu(B)). The heme-copper oxidases generally catalyze the four electron reduction of molecular oxygen in a sequence involving several intermediates. We have investigated the reaction of the fully reduced ba(3) with O(2) using stopped-flow techniques. Transient visible absorption spectra indicated that a fraction of the enzyme decayed to the oxidized state within the dead time (~1ms) of the stopped-flow instrument, while the remaining amount was in a reduced state that decayed slowly (k=400s(-1)) to the oxidized state without accumulation of detectable intermediates. Furthermore, no accumulation of intermediate species at 1ms was detected in time resolved resonance Raman measurements of the reaction. These findings suggest that O(2) binds rapidly to heme a(3) in one fraction of the enzyme and progresses to the oxidized state. In the other fraction of the enzyme, O(2) binds transiently to a trap, likely Cu(B), prior to its migration to heme a(3) for the oxidative reaction, highlighting the critical role of Cu(B) in regulating the oxygen reaction kinetics in the oxidase superfamily.
authors.affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY, USA.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Egawa
authors.forename: Ying
authors.initials: Y
authors.lastname: Chen
authors.forename: James A
authors.initials: JA
authors.lastname: Fee
authors.forename: Syun-Ru
authors.initials: SR
authors.lastname: Yeh
authors.forename: Denis L
authors.initials: DL
authors.lastname: Rousseau
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D003573:Cytochrome b Group / Q000737:chemistry / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013059:Spectrum Analysis, Raman; D016963:Thermus thermophilus / Q000201:enzymology* / Q000378:metabolism; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D003573:Cytochrome b Group; D005296:Ferrous Compounds; C027728:heme a; D006418:Heme; D003300:Copper; D007501:Iron; C058557:cytochrome ba3; D003576:Electron Transport Complex IV; D010100:Oxygen