Abstract

ABSTRACT: A range of conformationally distinct functional states within the T quaternary state of hemoglobin are accessed and probed using a combination of mutagenesis and sol-gel encapsulation that greatly slow or eliminate the T f R transition. Visible and UV resonance Raman spectroscopy are used to probe the proximal strain at the heme and the status of the R 1 2 interface, respectively, whereas CO geminate and bimolecular recombination traces in conjunction with MEM (maximum entropy method) analysis of kinetic populations are used to identify functionally distinct T-state populations. The mutants used in this study are Hb(N 102A) and the R99-R99 cross-linked derivative of Hb(W 37E). The former mutant, which binds oxygen noncooperatively with very low affinity, is used to access low-affinity ligated T-state conformations, whereas the latter mutant is used to access the high-affinity end of the distribution of T-state conformations. A pattern emerges within the T state in which ligand reactivity increases as both the proximal strain and the R 1 2 interface interactions are progressively lessened after ligand binding to the deoxy T-state species. The ligation and effector-dependent interplay between the heme environment and the stability of the Trp 37 cluster in the hinge region of the R 1 2 interface appears to determine the distribution of the ligated T-state species generated upon ligand binding. A qualitative model is presented, suggesting that different T quaternary structures modulate the stability of different R dimer conformations within the tetramer. Allosteric transitions represent an important mechanism for regulating protein reactivity. Hemoglobin has been and continues to be the prototype for studying the molecular basis of allostery. For hemoglobin as well as for other allosteric proteins, well-characterized initial and final endpoint structures form the basis for most mechanistic models put forth to account for the observed allostery. The MWC two-state model (1), used to explain many of the salient aspects of allostery within the hemoglobin tetramer, was based largely on the premise that there are oxygenation-dependent changes in the equilibrium between two functionally distinct quaternary states: the low-affinity T quaternary state and the highaffinity R quaternary state (2-6). These states are correlated with the T-and R-quaternary structures derived primarily from X-ray crystallographic studies on two stable endpoint Hb species, deoxyHb and fully ligated Hb, respectively. A growing number of studies indicate that a detailed molecular understanding of allostery in Hb requires the consideration of more than two affinity states. X-ray crystallographic measurements (7) and NMR solution-phase studies (8) have shown that fully ligated hemoglobin manifests an ensemble, the R e ensemble, of energetically similar but spatially distinct quaternary structures. In contrast, several high-resolution crystallographic studies have shown that fully deoxygenated hemoglobin has virtually the same T quaternary structure under high-salt and low-salt crystallization conditions (9-11). Such a structural observation might reasonably be construed to imply that the T state should have a narrow range of ligand binding properties; however, this is not the case. The ligand binding properties of the T state show considerable variability in response to the presence of effectors (12-19), partial ligation (20, 21), partial replacement of the four heme irons with other metals