Linking conformation change to hemoglobin activation via chain-selective time-resolved resonance Raman spectroscopy of protoheme/mesoheme hybrids |
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Authors: | Gurusamy Balakrishnan Mohammed Ibrahim Piotr J Mak Jessica Hata James R Kincaid Thomas G Spiro |
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Institution: | (1) Department of Chemistry, University of Washington, Seattle, WA 98195, USA;(2) Department of Chemistry, Marquette University, Milwaukee, WI 53233, USA |
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Abstract: | Time-resolved resonance Raman (RR) spectra are reported for hemoglobin (Hb) tetramers, in which the α and β chains are selectively
substituted with mesoheme. The Soret absorption band shift in mesoheme relative to protoheme permits chain-selective recording
of heme RR spectra. The evolution of these spectra following HbCO photolysis shows that the geminate recombination rates and
the yields are the same for the two chains, consistent with recent results on 15N-heme isotopomer hybrids. The spectra also reveal systematic shifts in the deoxyheme ν
4 and ν
Fe–His RR bands, which are anticorrelated. These shifts are resolved for the successive intermediates in the protein structure,
which have previously been determined from time-resolved UV RR spectra. Both chains show Fe–His bond compression in the immediate
photoproduct, which relaxes during the formation of the first intermediate, Rdeoxy (0.07 μs), in which the proximal F-helix is proposed to move away from the heme. Subsequently, the Fe–His bond weakens, more
so for the α chains than for the β chains. The weakening is gradual for the β chains, but is abrupt for the α chains, coinciding
with completion of the R–T quaternary transition, at 20 μs. Since the transition from fast- to slow-rebinding Hb also occurs
at 20 μs, the drop in the α chain ν
Fe–His supports the localization of ligation restraint to tension in the Fe–His bond, at least in the α chains. The mechanism is
more complex in the β chains. |
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Keywords: | Protoheme/mesoheme Hybrid hemoglobin Resonance Raman Geminate recombination Allostery |
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