Accommodation of CO in the di-heme active site of cytochrome bd terminal oxidase from Escherichia coli

Abstract

Catalytic mechanisms of reduction of O2 to 2H2O by respiratory terminal oxidases have been extensively investigated. Tri-heme (b558, b595, d) cytochrome bd oxidases presumably utilize a dihemic site composed of high-spin hemes d and b595. 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 b595. This is the first clear evidence of interaction of heme b595 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 b595 bound to CO, heme d remains unliganded, hence the dihemic O2-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 b595 in 5–10% of the cytochrome. The equilibration process is possibly associated with an exchange of heme d endogenous ligand.