Low-Power Picosecond Resonance Raman Evidence for Histidine Ligation to Heme a 3 after Photodissociation of CO from Cytochrome c Oxidase Johannes P. M. Schelvis, ² Geurt Deinum, ²,§ Constantinos A. Varotsis, ‡ Shelagh Ferguson-Miller, ² and Gerald T. Babcock* ,² Contribution from the Department of Chemistry, LASER Laboratory, and Department of Biochemistry, Michigan State UniVersity, East Lansing, Michigan 48824, and Department of Chemistry, UniVersity of Crete, 71409, Iraklion, Crete, Greece ReceiVed December 2, 1996. ReVised Manuscript ReceiVed March 14, 1997 X Abstract: Several models have been proposed for the ligand dynamics in the heme a 3 2+ /Cu B 1+ binuclear pocket in cytochrome oxidase following CO photodissociation. These range from straightforward heme pocket relaxation to a variety of ligand exchange processes that have been proposed to be of relevance to the proton pumping function of the enzyme. To provide discrimination between these models, we have used picosecond time-resolved, pump- probe resonance Raman spectroscopy to study the photolysis process in the enzyme isolated from beef heart and from Rhodobacter sphaeroides. The intermediate observed within 5 ps of photolysis with low-energy probe pulses (10-20 nJ/pulse) is the high-spin, five-coordinate 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 low to avoid promoting any significant photochemistry during the spectral acquisition phase of the pump-probe experiment. From these observations, 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 a 3 site by means of a ligand exchange process can be ruled out. We observe similar picosecond transient resonance Raman spectra for the CO complex of Rb. sphaeroides cytochrome c oxidase. From these results and earlier time-resolved Raman and FTIR measurements, we propose a model for the relaxation dynamics of the heme a 3 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. Introduction Cytochrome c oxidase (CcO) is the terminal electron acceptor in the mitochondrial respiratory chain (see ref 1 for recent reviews). The enzyme contains four redox active metal sites: heme a, heme a 3 , Cu A , and Cu B . Molecular O 2 , which is the final electron acceptor, is bound and reduced at the a 3 /Cu B binuclear active site. In addition to its electron transfer and dioxygen reduction functions, cytochrome oxidase also functions as a proton pump and uses the free energy released during O 2 reduction to translocate protons across the mitochondrial membrane. Time-resolved spectroscopies have been used to study the reaction of O 2 with the enzyme and considerable insight into mechanism has resulted. 1b,c,f Nonetheless, it is sometimes difficult to study ligand binding and coordination chemistry in the binuclear center by using only O 2 as the ligating species, owing to its high reactivity with the enzyme (turnover rate about 1000 electron s -1 ). Other exogenous ligands such as CO, CN - , and NO often can be used more effectively for this purpose. CO, in particular, which forms a stable complex with the fully reduced enzyme, 2 has been used in time-resolved experiments to investigate the relaxation kinetics at the active site after its photodissociation. The relevance of such measurements has become especially clear recently, as distal ligand exchange mechanisms for coupling proton translocation to redox and coordination events in the binuclear center are currently under active investigation. 3,4 Alben and co-workers 5 showed that CO transiently binds to Cu B after it has been photodissociated from heme a 3 . Woodruff and co-workers 6 subsequently performed an incisive and detailed study of the kinetics of CO photodissociation. From their results, it is clear that CO dissociates from heme a 3 within 100 ² Michigan State University. ‡ University of Crete. § Present address: G. R. Harrison Spectroscopy Laboratory, Massa- chusetts Institute of Technology, Cambridge, MA 02139. * To whom correspondence should be addressed. 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