Fast Deuterium Access to the Buried Magnesium/Manganese Site in CytochromecOxidase

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Abstract
The recently determined crystal structures of bacterial and bovine cytochrome c oxidases show an area of organized water within the protein immediately above the active site where oxygen chemistry occurs. A pathway for exit of protons or water produced during turnover is suggested by possible connections of this aqueous region to the exterior surface. A non-redox-active Mg2+ site is located in the interior of this region, and our previous studies [Florens, L., Hoganson, C., McCracken, J., Fetter, J., Mills, D., Babcock, G. T., and Ferguson-Miller, S. (1998) in Phototropic Prokaryotes (Peschek, G. A., Loeffelhard, W., and Schmetterer, G., Eds.) Kluwer Academic/Plenum, New York] have shown that the protons of water molecules that coordinate the metal can be exchanged within minutes of mixing with 2H2O. Here we examine the extent and rate of deuterium exchange, using a combination of rapid freeze−quench and electron spin echo envelope modulation (ESEEM) analysis of Mn2+-substituted cytochrome c oxidase, which retains full activity. In the oxidized enzyme at room temperature, deuterium exchange at the Mn2+ site occurs in less than 11 ms, which corresponds to an apparent rate constant higher than 3000 s-1. The extent of deuterium substitution is dependent on the concentration of 2H2O in the sample, indicative of rapid equilibrium, with three inner sphere 2H2O exchanged per Mn2+. This indicates that the water ligands of the Mn2+/Mg2+ site, or the protons of these waters, can exchange with bulk solvent at a rate consistent with a role for this region in product release during turnover.