Subunit-Selective Interrogation of CO Recombination in Carbonmonoxy Hemoglobin by Isotope-Edited Time-Resolved Resonance Ranian Spectroscopy.

ABSTRACT: Hemoglobin (Hb) is an allosteric tetrameric protein made up of αβ heterodimers. The a and ,3 chains are similar, but are chemically and structurally distinct. To investigate dynamical differences between the chains, we have prepared tetramers in which the chains are isotopically distinguishable, via reconstitution with [sup15]N-heme. Ligand recombination and heme structural evolution, following HbCO dissociation, was monitored with ëhain selectivity by resonance Raman (RR) spectrcoscopy. For α but not for β chains, the frequency of the v[sub4] porphyrin breathing mode increased on the microsecond time scale. This increase is a manifestation ofproximal tension in the Hb T-state, and its time course is parallel to the formation of T contacts, as determined previously by UVRR spectroscopy. Despite the localization of proximal constraint in the a chains, geminate recombination was found to be equally probable in the two chains, with yields of 39 ± 2%. We discuss the possibility that this equivalence is coincidental, in the sense that it arises from the evolutionary pressure for cooperativity, or that it reflects mechanical coupling across the afi interface, evidence for which has emerged from UVRR studies of site mutants. [ABSTRACT FROM AUTHOR]