The X-ray structures of bacterial and mammalian cytochrome oxidases reveal possible pathways to facilitate the translocation of protons from the electronegative side of the membrane to the heme-copper bimetallic center and extending across the membrane. Site-directed mutants of the cytochrome oxidase from Rhodobacter sphaeroides have been used to examine the functions of these pathways. Recent results from our laboratory and from collaborators will be summarized. One of the putative channels, the K-channel, is disabled by the K362M mutation. Single-turnover kinetics have been performed in conjunction with the laboratories of Dr. Alexander Konstantinov (Moscow State University), Dr. Peter Brzezinski (University of Göteborg) and Dr. Francis Milieu (University of Arkansas). The data indicate that the K362M mutation blocks the initial reduction of the heme-copper bimetallic center, perhaps by preventing the protonation of one or more sites necessary to stabilize the reduced meial centers. The kinetics of the steps following this appear not to be inhibited by this mutation. Consistent with this is the observation that the K362M mutant has cytochrome c peroxidase activity. The use of hydrogen peroxide in place of dioxygen as an oxidant bypasses the need to pre-reduce the heme-copper center prior to the formation of the peroxy intermediate. In contrast, the D-channel, which is disabled by either the E286Q or D132N mutations, is required for steps in the catalytic cycle following the interaction of the reduced heme-copper center with oxygen. The experiments indicate that the K-channel and the D-channel are operational at different steps in the catalytic cycle. (Supported by the NIH).
|Original language||English (US)|
|State||Published - 1997|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology