ENDOR AND ESEEM STUDIES OF CYTOCHROME-C-OXIDASE - EVIDENCE FOR EXCHANGEABLE PROTONS AT THE CU(A) SITE

Electron nuclear double resonance (ENDOR) and electron spin echo envel ope modulation (ESEEM) spectroscopies were used to study whether proto ns in the immediate protein environment around CU(A) in cytochrome c o xidase are susceptible to solvent exchange. The enzyme was incubated i n buffered D2O under resting or turnover conditions for 90 min and the n frozen to quench the hydrogen/deuterium-exchange process. ENDOR spec tra of the deuterated sample were essentially identical to those of co ntrol samples. The ESEEM spectra, however, provided a clear indication of the introduction of deuterium into the CU(A) environment following incubation in buffered D2O. The extent of deuterium incorporation was not affected by enzyme turnover. An analysis of the ESEEM data indica ted that water is in reasonably close proximity to the CU(A) site, but not in the immediate coordination sphere of the metal(s). We estimate a minimum distance of 5.4 angstrom between the CU(A) center and the p rotein/water interface. This relatively short surface separation dista nce is consistent with the role of CU(A) as the immediate oxidant of c ytochrome c in the cytochrome oxidase (Hill, B. C. (I 99 1) J. Biol. C hem. 266, 2219-2226).