Kinetic analysis of the reaction of cytochrome cc' with carbon monoxide.

The equilibria and kinetics of the reaction of the diheme proteins cytochrome cc’ derived from Rhodospirillum rubrum and Chromatium with carbon monoxide have been examined. There are wide differences in many respects. The pigment from Chromatium is half saturated by 0.2 pM CO, while that of R. rubrum requires a concentration of 400 PM. The kinetics of the combination reaction also differs. In stopped flow experiments, the rate for R. rubrum cytochrome cc’ is independent of CO concentration over the range examined, while that for the Chromatium pigment is proportional to CO concentration and yielded a second order rate constant of 260 M-’ set-1 at room temperature. Flash photochemical experiments gave biphasic recombination reactions, with rates independent of carbon monoxide concentration in both cases. The reactions are much faster than those observed by the stopped flow method. The velocity of dissociation of CO was measured for the pigment from R. rubrum by dilution and was 0.009 set-I, at 22' and pH 7.4. The equilibrium between CO and both proteins could be displaced by steady illumination from medium intensity sources such as tungsten filaments. In the Chromatium pigment, the rates of photodissociation and recombination were independent of light intensity. They varied with CO concentration and were numerically equal at CO concentrations greater than 1 X ~O+M. In R. rubrum cytochrome cc’ the photochemical behavior was analogous to that of myoglobin in that there was the usual dependence of rate of dissociation on light intensity. Preliminary experiments on the reaction of the reduced hemoproteins with oxygen and on the reaction of the carbon monoxide complexes with oxygen have given complicated results which cannot be summarized briefly. The findings may be explained by the hypothesis that a bound carbon monoxide molecule, after dissociation from the heme as judged spectrophotometrically, retains an association with the protein molecule. The conclusions resulting from this scheme are discussed in detail.