Binding of O2 and Its Reduction Are Both Retarded by Replacement of Valine 279 by Isoleucine in Cytochrome c Oxidase from Paracoccus denitrificans

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Abstract
The crystal structure of the heme−copper oxidases suggested a putative channel of oxygen entry into the heme−copper site of O2 reduction. Changing a conserved valine near this center in cytochrome bo3 of Escherichia coli to isoleucine caused a significant increase in the apparent KM for oxygen with little or no change in Vmax, suggesting that oxygen diffusion had been partially blocked [Riistama, S., Puustinen, A., García-Horsman, A., Iwata, S., Michel, H., and Wikström, M. (1996) Biochim. Biophys. Acta1275, 1−4]. To study this phenotype further using rapid kinetic methods, the corresponding change (V279I) has been made in cytochrome aa3 from Paracoccus denitrificans. In this mutant, the apparent KM for oxygen is 8 times higher than in the wild-type enzyme, whereas Vmax is decreased only to approximately half of the wild-type value. Flow-flash kinetic measurements show that the initial binding of oxygen to the heme of the binuclear site is indeed much slower in the mutant than in the wild-type enzyme. However, the subsequent phases of the reaction with O2 are also slow although the pure heme-to-heme electron transfer process is essentially unperturbed. It is suggested that the mutation sterically hinders O2 entry into the binuclear site and that it may also perturb the structure of local water molecules involved in proton transfer to this site.