Binding of O sub(2) and Its Reduction Are Both Retarded by Replacement of Valine 279 by Isoleucine in Cytochrome c Oxidase from Paracoccus denitrificans

• Published in: Biochemistry (Easton) 2000-05, Vol.39 (21), p.6465-6372
• Main Authors: Riistama, S, Puustinen, A, Verkhovsky, MI, Morgan, JE, Wikstroem, M
• Format: Article
• Language: English
• Subjects: cytochrome aa3; cytochrome bo3; Paracoccus denitrificans
• ISSN: 0006-2960

The crystal structure of the heme-copper oxidases suggested a putative channel of oxygen entry into the heme-copper site of O sub(2) reduction. Changing a conserved valine near this center in cytochrome bo sub(3) of Escherichia coli to isoleucine caused a significant increase in the apparent K sub(M) for oxygen with little or no change in V sub(max), suggesting that oxygen diffusion had been partially blocked [Riistama, S., Puustinen, A., Garcia-Horsman, A., Iwata, S., Michel, H., and Wikstroem, M. (1996) Biochim. Biophys. Acta 1275, 1-4]. To study this phenotype further using rapid kinetic methods, the corresponding change (V279I) has been made in cytochrome aa sub(3) from Paracoccus denitrificans. In this mutant, the apparent K sub(M) for oxygen is 8 times higher than in the wild-type enzyme, whereas V sub(max) 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 O sub(2) are also slow although the pure heme-to-heme electron transfer process is essentially unperturbed. It is suggested that the mutation sterically hinders O sub(2) entry into the binuclear site and that it may also perturb the structure of local water molecules involved in proton transfer to this site.