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

Abstract

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.