Early photolysis intermediates of native bovine rhodopsin (RHO) are investigated by nanosecond laser photolysis near physiological temperature. Absorption difference spectra are collected after excitation with 477-, 532-, and 560-nm laser pulses of various energies and with 477-nm laser excitation at 5, 12, 17, 21, and 32 degrees C. The data are analyzed by using singular-value decomposition (SVD) and a global exponential fitting routine. Two rate constants associated with distinct spectral changes are observed during the time normally associated with the decay of bathorhodopsin to lumirhodopsin. Various models consistent with this observation are considered. A sequential model in which there is a reversible step between a bathorhodopsin intermediate and a new intermediate (BSI), which is blue-shifted relative to lumirhodopsin, is shown to best fit the data. The temperature dependence of the observed and calculated rate constants leads to linear Arrhenius plots. Extrapolation of the temperature dependence suggests that BSI should not be observable after rhodopsin photolysis at temperatures below -100 degrees C. The results are discussed with regard to the artificial visual pigments cis-5,6-dihydroisorhodopsin and 13-demethylrhodopsin. It is proposed that the rate of the BATHO to BSI transition is limited by the relaxation of the strained all-trans-retinal chromophore within a tight protein environment. The transition to LUMI involves chromophore relaxation concurrent with protein relaxation. While the first process is strongly affected by changes in the chromophore, the second transition seems to be determined more by protein relaxation.

A previously undescribed spectrokinetic assay for the entry of water into the distal heme pocket of wild-type and mutant myoglobins is presented. Nanosecond photolysis difference spectra were measured in the visible bands of sperm whale myoglobin as a function of distal pocket mutation and temperature. A small blue shift in the 560-nm deoxy absorption peak marked water entry several hundred nanoseconds after CO photodissociation. The observed rate suggests that water entry is rate-limited by the escape of internal dissociated CO. The heme pocket hydration and geminate recombination yields were found to be the primary factors controlling the overall bimolecular association rate constants for CO binding to the mutants studied. The kinetic analysis provides estimates of 84%, 60%, 40%, 0%, and 99% for the steady-state hydrations of wild-type, H64Q, H64A, H64L, and V68F deoxymyoglobin, respectively. The second-order rate constants for CO and H 2O entry into the empty distal pocket of myoglobin are markedly different, 8 ؋ 10 7 and 2 ؋ 10 5 M ؊1 ⅐s ؊1 , respectively, suggesting that hydrophobic partitioning of the apolar gas from the aqueous phase into the relatively apolar protein interior lowers the free energy barrier for CO entry.distal water occupancy ͉ spectrokinetic assay ͉ Mb mutants ͉ rebinding kinetics

Time-resolved electronic absorption, infrared, resonance Raman, and magnetic circular dichroism spectroscopies are applied to characterization of the intermediate that is formed within 20 ps after photodissociation of CO from cytochrome a3 in reduced cytochrome oxidase. This intermediate decays with the same half-life (1l is) as the postphotodissociation Cu'-CO species previously observed by time-resolved infrared. The transient UV/visible spectra, kinetics, infrared, and Raman evidence suggest that an endogenous ligand is transferred from CUB to Fe,3 when CO binds to CUB, forming a cytochrome a3 species with axial ligation that differs from the reduced unliganded enzyme. The timeresolved magnetic circular dichroism results suggest that this transient is high-spin and, therefore, five-coordinate. Thus we infer that the ligand from CUB binds on the distal side of cytochrome a3 and displaces the proximal histidine imidazole. This remarkable mechanistic feature is an additional aspect of the previously proposed "ligand-shuttle" activity of the CuB/Fe,3 pair. We speculate as to the identity ofthe ligand that is transferred between CUB and Fe,13 and suggest that the ligand shuttle may play a functional role in redox-linked proton translocation by the enzyme.In a recent time-resolved infrared (TRIR) study (1) of the events after photodissociation of CO from cytochrome (cyt) a3 of reduced beef heart cytochrome oxidase (CcO), we reported conclusive evidence that photodissociated CO binds quantitatively to CuB at room temperature prior to equilibrating with solution. In a parallel kinetics study (6.E., P. M.