Allosteric intermediates in hemoglobin. 1. Nanosecond time-resolved circular dichroism spectroscopy.

Time-resolved circular dichroism (TRCD) studies performed on photolyzed hemoglobin-CO complex (HbCO) probe room temperature protein relaxations in Hb, including the R --> T allosteric transition. TRCD spectroscopy of photolysis intermediates in the near-UV (250-400 nm) spectral region provides a diagnostic for T-like structure at the alpha 1 beta 2 interface via the effect of quaternary structure on the UV CD of aromatic residues. The TRCD of porphyrin-based transitions in the UV and Soret regions, reflecting transition-dipole couplings between hemes and aromatic residues over a radius wide enough to permit heme-interface and inter-dimer interactions, is modulated by the tertiary and quaternary structure of photolysis intermediates. In the allosteric core model of Hb cooperativity, Fe-CO bond breakage initiates a heme structural change, thought to be heme doming, that is transmitted to the alpha 1 beta 2 interface via the F helix. The TRCD results, analyzed in light of kinetic information from time-resolved absorption studies, suggest specific features for the mechanism by which the ensuing tertiary and quaternary conformational changes propagate through the protein. In particular, the UV-TRCD indicates that the alpha 1 beta 2 interface responds within several hundred nanoseconds to initial events at the heme by shifting from an R toward a T-like interface. The appearance of T-like character at the alpha 1 beta 2 interface tens of microseconds before the appearance of equilibrated T state deoxyHb indicates that the R --> T transition in photolyzed HbCO is a stepwise process, as previously suggested by time-resolved resonance Raman studies.

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-microseconds R --> T quaternary transition and a previously unassigned 1-microseconds intraprotein relaxation. On the basis of the modeled intermediate spectra, as well as UV circular dichroism results observed on this time scale [Björling, S.C., Goldbeck, R.A., Paquette, S.J., Milder, S.J., & Kliger, D.S. (1996) Biochemistry 35, 8619-8627], the 1-microsecond 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.

Time-resolved circular dichroism and absorption studies of the photolysis reaction of (carbonmonoxy)myoglobin.

Time-resolved circular dichroism (TRCD) and absorption spectroscopy are used to follow the photolysis reaction of (carbonmonoxy)myoglobin (MbCO). Following the spectral changes associated with the initial loss of CO, a subtle change is observed in the visible absorption spectrum of the Mb product on a time scale of a few hundred nanoseconds. No changes are seen in the CD spectrum of Mb in the visible and near-UV regions subsequent to the loss of CO. The data suggest the existence of an intermediate found after ligand loss from MbCO that is similar in structure to the final Mb product.