ALLOSTERIC INTERMEDIATES IN HEMOGLOBIN .1. NANOSECOND TIME-RESOLVED CIRCULAR-DICHROISM SPECTROSCOPY

Time-resolved circular dichroism (TRCD) studies performed on photolyze d hemoglobin-CO complex (HbCO) probe room temperature protein relaxati ons in Hb, including the R --> T allosteric transition. TRCD spectrosc opy of photolysis intermediates in the near-UV (250-400 nm) spectral r egion 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 ar omatic residues, The TRCD of porphyrin-based transitions in the UV and Sorer regions, reflecting transition-dipole couplings between hemes a nd aromatic residues over a radius wide enough to permit heme-interfac e and inter-dimer interactions, is modulated by the tertiary and quate rnary structure of photolysis intermediates. In the allosteric core mo del of Hb cooperativity, Fe-CO bond breakage initiates a heme structur al 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 l ight of kinetic information from time-resolved absorption studies, sug gest 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) interfa ce responds within several hundred nanoseconds toinitial 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 microsec onds before the appearance of equilibrated T state deoxyHb indicates t hat the R --> T transition in photolyzed HbCO is a stepwise process, a s previously suggested by time-resolved resonance Raman studies.