Journal
BIOCHEMISTRY
Volume 45, Issue 9, Pages 2820-2835Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi050010i
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Funding
- NHLBI NIH HHS [P01 HL051084, P01 HL071064] Funding Source: Medline
- NIBIB NIH HHS [R01 EB-00296, R01 EB000296] Funding Source: Medline
- NIGMS NIH HHS [P01 GM058890, P01 GM-58890] Funding Source: Medline
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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 -> R transition. Visible and UV resonance Raman spectroscopy are used to probe the proximal strain at the heme and the status of the alpha(1)beta(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 beta 102A) and the alpha 99-alpha 99 cross-linked derivative of Hb(W beta 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 alpha(1)beta(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 beta 37 cluster in the hinge region of the alpha(1)beta(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 alpha beta dimer conformations within the tetramer.
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