title: Accommodation of CO in the di-heme active site of cytochrome bd terminal oxidase from Escherichia coli.
abstract: Catalytic mechanisms of reduction of O(2) to 2H(2)O by respiratory terminal oxidases have been extensively investigated. Tri-heme (b(558), b(595), d) cytochrome bd oxidases presumably utilize a dihemic site composed of high-spin hemes d and b(595). We performed a CO photolysis/recombination study of the purified fully reduced cytochrome bd from Escherichia coli. Spectrum of CO photolysis suggests photodissociation of the ligand from heme d and from part of heme b(595). This is the first clear evidence of interaction of heme b(595) with CO at room temperature. The amount of the heme d-CO species is higher after recombination than before photolysis. In the enzyme population with heme b(595) bound to CO, heme d remains unliganded, hence the dihemic O(2)-reducing pocket in cytochrome bd can bind one rather than two diatomic molecules. Occupancy of the site by one ligand molecule probably blocks access of a second molecule. Thus cytochrome bd exhibits strong negative cooperativity in ligand binding. Immediately after photolysis/recombination CO occupies 100% of the heme d sites, whereas after equilibration, the ligand gets located at heme d in 90-95% and at heme b(595) in 5-10% of the cytochrome. The equilibration process is possibly associated with an exchange of heme d endogenous ligand.
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russian Federation. bor@genebee.msu.su
authors.forename: Vitaliy B
authors.initials: VB
authors.lastname: Borisov
authors.forename: Michael I
authors.initials: MI
authors.lastname: Verkhovsky
mesh_terms: D002248:Carbon Monoxide / Q000737:chemistry*; D020134:Catalytic Domain; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry*; D045222:Electron Transport Chain Complex Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D010088:Oxidoreductases / Q000737:chemistry*; D010782:Photolysis; D011485:Protein Binding
chemical_list: D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D006418:Heme; D002248:Carbon Monoxide; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli