CONFORMATIONAL RELAXATION AND LIGAND-BINDING IN MYOGLOBIN

Absorption spectroscopy with nanosecond time resolution shows that myo globin undergoes conformational relaxation on the same time scale as g eminate rebinding of carbon monoxide. Ligand rebinding following photo dissociation of the heme-CO complex was measured from the amplitude of the average difference spectrum, while conformational changes were me asured from changes in the detailed shape of the Soret spectra of the deoxyhemes. Experiments in which the solvent viscosity was varied betw een 1 and 300 cP and the temperature between 268 and 308 K were analyz ed by fitting the multiwavelength kinetic data with both empirical and molecular models. Novel numerical techniques were employed in fitting the data, including the use of singular value decomposition to remove the effects of temperature and solvent on the spectra and of a Monte Carlo method to overcome the multiple minimum problem in searching par ameter space. The molecular model is the minimal model that incorporat es all of the major features of myoglobin kinetics at ambient temperat ures, including a fast and slow rebinding conformation and two geminat e states for each conformation. The results of fitting the kinetic dat a with this model indicate that the geminate-rebinding rates for the t wo conformations differ by at least a factor of 100. The differences b etween the spectra of the two conformations generated from the fits ar e similar to the differences between those of the R and T conformation s of hemoglobin. In modeling the data, the dependence of the rates on temperature and viscosity was parametrized using a modification of Kra mers theory which includes the contributions of both protein and solve nt to the friction. The rate of the transition from the fast to the sl ow rebinding conformation is found to be inversely proportional to the viscosity when the viscosity exceeds about 30 cP and nearly viscosity independent at low viscosity. The viscosity dependence at high viscos ities suggests that the two conformations differ by the global displac ement of protein atoms on the proximal side of the heme observed by X- ray crystallography. We suggest that the conformational change observe d in our experiments corresponds to the final portion of the nonexpone ntial conformational relaxation recently observed by Anfinrud and co-w orkers, which begins on a picosecond time scale. Furthermore, extrapol ation of our data to temperatures near that of the solvent glass trans ition suggests that this conformational relaxation may very well be th e one postulated by Frauenfelder and co-workers to explain the decreas e in the rate of geminate rebinding with increasing temperature above 180 K.