Stopped-flow, laser-flash photolysis studies on the reactions of CO and O2 with the cytochrome caa3 complex from Bacillus subtilis: conservation of electron transfer pathways from cytochrome c to O2.
The reaction of CO and O2 with fully reduced cytochrome caa3 from Bacillus subtilis has been studied by rapid reaction spectrophotometry. The fully reduced caa3 complex reacts with CO to give a spectrum that is characteristic of formation of ferrocytochrome a3-CO. This adduct is photosensitive, and its recombination rate is proportional to CO concentration with a bimolecular value of 1.2 x 10(5)M-1 s-1. When the CO compound of the reduced complex is exposed to O2, the rate of oxidation proceeds at 0.1 s-1, which is assigned as the CO off rate. These kinetic constants give an equilibrium dissociation constant for the CO complex of 0.83 microM. Photolysis of the CO adduct in the presence of O2 reveals three reaction phases over the first 3 ms and an additional phase on the second time scale. A kinetic model is proposed in which fully reduced oxidase first combines with O2 and then electron transfer commences from both cytochrome a and a3, followed rapidly by electron input from CuA and the cytochrome c domain. An equivalent kinetic model has been used to account for the reactivity of mammalian cytochrome c oxidase in its electrostatic complex with soluble cytochrome c [Hill, B. C., (1994) J. Biol. Chem. 269, 2419-2425]. However, unlike the mitochondrial complex, the reactivity of cytochrome c in the B. subtilis caa3 complex is unaffected by ionic strength. Thus the cytochrome c moiety in the B. subtilis caa3 complex seems to be fixed in a reactive orientation by its covalent association with the rest of the oxidase complex. The pathway of electron transfer from cytochrome c to O2 appears very well conserved from B. subtilis to the mammalian respiratory chain, making the B. subtilis protein a good model to probe intersite electron transfer within the cytochrome c-cytochrome oxidase complex.
Hill
B C
BC
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
D001412:Bacillus subtilis / Q000201:enzymology*; D002248:Carbon Monoxide / Q000737:chemistry*; D003574:Cytochrome c Group / Q000378:metabolism*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D007834:Lasers; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D010782:Photolysis
D003574:Cytochrome c Group; D002248:Carbon Monoxide; D003576:Electron Transport Complex IV; D010100:Oxygen