Fast deuterium access to the buried magnesium/manganese site in cytochrome c oxidase

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 (H2O)-H-2. 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 (H2O)-H-2 in the sample, indicative of rapid equilibrium, with three inner sphere (H2O)-H-2 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.