Allosteric intermediates in hemoglobin .2. Kinetic modeling of HbCO photolysis

Nanosecond absorption spectra are measured in the Soret and near-UV spectral regions of human hemoglobin (Hb) after laser photolysis of the carbonyl adduct in order to study the dynamics of globin tertiary and quaternary conformational changes. Spectra and concentrations of physical intermediates, distinguished by extent of heme ligation and intraprotein relaxation, are obtained from a global analysis using a microscopic kinetic model that explicitly accounts for six observed relaxation and recombination processes. Three observed rate constants for CO rebinding and two intraprotein relaxation constants obtained are similar to constants determined by Hofrichter et al. [(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2235], the latter two comprising the 20-30-mu s R --> T quaternary transition and a previously unassigned 1-mu s intraprotein relaxation. On the basis of the modeled intermediate spect-ra, as well as UV circular dichroism results observed on this time scale [Bjorling, S. C., Goldbeck, R. A., Paquette, S. J., Milder, S. J., & Kliger, D. S. (1996) Biochemistry 35, 8619-8627], the 1-mu s relaxation is assigned to heme conformational changes concomitant with a relaxation of protein conformation at the alpha 1 beta 2 interface corresponding to an initial step in a compound R --> T reaction path.