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Abstract

he effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochromec oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a α-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochromec yielded a kinetic difference spectrum resembling that of heme a₃. This observation, and the absence of transient absorbance changes 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 hemea but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where Δψ generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.