STOPPED-FLOW, LASER-FLASH PHOTOLYSIS STUDIES ON THE REACTIONS OF CO AND O-2 WITH THE CYTOCHROME CAA(3) COMPLEX FROM BACILLUS SUBTILIS - CONSERVATION OF ELECTRON TRANSFER PATHWAYS FROM CYTOCHROME C TO O-2

摘要

The reaction of CO and O-2 With fully reduced cytochrome caa(3) from Bacillus subtilis has been studied by rapid reaction spectrophotometry. The fully reduced caa(3) complex reacts with CO to give a spectrum that is characteristic of formation of ferrocytochrome a(3)-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 O-2, 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 mu M Photolysis of the CO adduct in the presence of O-2 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 O-2 and then electron transfer commences from both cytochrome a and a(3), followed rapidly by electron input from Cu-A 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 caa(3) complex is unaffected by ionic strength. Thus the cytochrome c moiety in the B. subtilis caa(3) 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 O-2 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.