LOW-POWER PICOSECOND RESONANCE RAMAN EVIDENCE FOR HISTIDINE LIGATION TO HEME A(3) AFTER PHOTODISSOCIATION OF CO FROM CYTOCHROME-C-OXIDASE

Several models have been proposed for the ligand dynamics in the heme a(3)(2+)/CuB1+ binuclear pocket in cytochrome oxidase following CO; ph otodissociation. These range from straightforward heme pocket relaxati on to a variety of ligand exchange processes that have been proposed t o be of relevance to the proton pumping function of the enzyme. To pro vide discrimination between these models, we have used picosecond time -resolved, pump-probe resonance Raman spectroscopy to study the photol ysis process in the enzyme isolated from beef heart and from Rhodobact er sphaeroides. The intermediate observed within 5 ps of photolysis wi th low-energy probe pulses (10-20 nJ/pulse) is the high-spin, five-coo rdinate heme a(3)(2+) to which a histidine is ligated, as indicated by the observation of the Fe-His vibration at 220 cm(-1) Several control experiments demonstrate that the probe pulse energy is sufficiently l ow to avoid promoting any significant photochemistry during the spectr al acquisition phase of the pump-probe experiment. From these observat ions, we conclude that histidine is ligated to high-spin heme a(3)(2+) on the picosecond time scale following photolysis. Since H376 is the proximal a(3)(2+) ligand in the CO complex, our results indicate that this proximal ligation survives photolysis and that the control of the access of exogenous ligands to the heme as site by means of a ligand exchange process can be ruled out. We observe similar picosecond trans ient resonance Raman spectra for the CO complex of Rb. sphaeroides cyt ochrome c oxidase. From these results and earlier time-resolved Raman and FTIR measurements, we propose a model for the relaxation dynamics of the heme as pocket that involves picosecond migration of CO to the Cu-B center and relaxation of the a(3)(2+)-proximal histidine bond on the microsecond time scale following CO photolysis.