Mutation of Arg-54 strongly influences heme composition and rate and directionality of electron transfer in Paracoccus denitrificans cytochrome c oxidase

The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optic al spectroscopy, electrochemical and rapid kinetics techniques, and time-re solved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a alpha-band by 15 nm and partial occupation o f the low-spin heme site by heme O. Additionally, there was a marked decrea se in the midpoint potential of the low-spin heme, resulting in slow reduct ion of this heme species. A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a(3). This observation, and the absence of transient absorbance chang es at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu-A to the binuclear center may not occur via heme a but that instead direct electron transfer to the high- spin heme is the dominating process. This was supported by charge transloca tion measurements where Delta psi generation was completely inhibited in th e presence of HCN. Our results thus provide an example for how the interpla y between protein and cofactors can modulate the functional properties of t he enzyme complex.