Low-power picosecond resonance Raman evidence for histidine ligation to heme a3 after photodissociation of CO from cytochrome c oxidase

Several models have been proposed for the ligand dynamics in the heme a32+/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 a32+ 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 a32+ on the picosecond time scale following photolysis. Since H376 is the proximal a32+ 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 a3 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 as pocket that involves picosecond migration of CO to the Cu(B) center and relaxation of the a32+-proximal histidine bond on the microsecond time scale following CO photolysi

CO photolysis of cytochrome oxidase investigated by ps resonance Raman spectroscopy

Low-power picosecond resonance Raman spectroscopy was used to investigate the identity of the axial ligand of heme a3 and relaxation processes in the heme a3 pocket of cytochrome oxidase after CO photolysis. Our results show that the proximal histidine remains ligated to heme a3 after CO photolysis excluding the transient ligation of a photolabile, endogenous ligand. Furthermore, the relaxation of the heme a3 macrocycle modes occurs on the sub ps time scale, while relaxation of the heme pocket to its equilibrium conformation takes place on the μs time scal

CO Photolysis of Cytochrome Oxidase Investigated by Ps Resonance Raman Spectroscopy

Low-power picosecond resonance Raman spectroscopy was used to investigate the identity of the axial ligand of heme a3 and relaxation processes in the heme a3 pocket of cytochrome oxidase after CO photolysis. Our results show that the proximal histidine remains ligated to heme a3 after CO photolysis excluding the transient ligation of a photolabile, endogenous ligand. Furthermore, the relaxation of the heme a3 macrocycle modes occurs on the sub ps time scale, while relaxation of the heme pocket to its equilibrium conformation takes place on the μs time scale

Low-Power Picosecond Resonance Raman Evidence for Histidine Ligation to Heme A3 After Photodissociation of CO from Cytochrome C Oxidase

Several models have been proposed for the ligand dynamics in the heme a32+/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 a32+ 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 a32+ on the picosecond time scale following photolysis. Since H376 is the proximal a32+ 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 a3 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 as pocket that involves picosecond migration of CO to the Cu(B) center and relaxation of the a32+-proximal histidine bond on the microsecond time scale following CO photolysis

Ligand Dynamics in the Binuclear Site in Cytochrome Oxidase

The dioxygen-reduction mechanism in cytochrome oxidase relies on proton control of the electron-transfer events that drive the process. Recent work on proton delivery and efflux channels in the protein that are relevant to substrate reduction and proton pumping is considered, and the current status of this area is summarized. Carbon monoxide photo dissociation and the ligand dynamics that occur subsequent to photolysis have been valuable tools in probing possible coupling schemes for linking exergonic electron-transfer chemistry to endergonic proton translocation. Our picosecond-time-resolved Raman results show that the heme a3- proximal histidine bond remains intact following CO photo dissociation but that the local environment around the heme a3 center in the photoproduct is in a nonequilibrium state. This photoproduct relaxes to its equilibrium configuration on the same time scale as ligand release occurs from CUB' which suggests a coupling between the two events and a potential signaling pathway between the site of O2 binding and reduction and the putative element, CUB' that links the redox chemistry to the proton pump