title: The gateway to the active site of heme-copper oxidases.
abstract: The spectroscopy and dynamics of CO binding were measured for wild-type and mutant cytochromes bo, members of the superfamily of heme-copper oxidases. The results suggest that access of ligands, including substrate O2, to the binuclear Fe-Cu active site is controlled at two levels. CO recombination to the wild-type ubiquinol oxidase exhibited saturation kinetics (kmax = 190 s-1, Km = 2.4 mM), indicative of the existence of an intermediate in the ligand-binding pathway. FTIR spectroscopy and TRIR spectroscopy were used to demonstrate conclusively that this intermediate was a CuB-CO complex. Two mutant oxidases (His333Leu, His334Leu) which lack CuB showed no evidence of saturation of CO rebinding, even up to 21 mM CO. Also, the absolute rates of CO binding to the mutant oxidases were much greater than for wild type, even at CO concentrations well below the apparent Km for wild-type enzyme. These results clearly indicate that the copper ion at the binuclear site acts as an obligatory way station, or gate, severely limiting the approach of ligands to the heme active site. Further, an analysis of the rate constants for CO binding to CuB suggests that the protein structure external to the binuclear site regulates ligand entry into this site. We propose that these control mechanisms for substrate binding are operative throughout this general class of enzymes.
authors.affiliation: Los Alamos National Laboratory, New Mexico 87545.
authors.forename: D D
authors.initials: DD
authors.lastname: Lemon
authors.forename: M W
authors.initials: MW
authors.lastname: Calhoun
authors.forename: R B
authors.initials: RB
authors.lastname: Gennis
authors.forename: W H
authors.initials: WH
authors.lastname: Woodruff
mesh_terms: D001665:Binding Sites; D003300:Copper; D003573:Cytochrome b Group; D003580:Cytochromes / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006420:Hemeproteins / Q000378:metabolism*; D007700:Kinetics; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D006420:Hemeproteins; D003300:Copper; C017168:cytochrome bo, E coli