Subunit-selective interrogation of CO recombination in carbonmonoxy hemoglobin by isotope-edited time-resolved resonance Raman spectroscopy.
Subunit-selective interrogation of CO recombination in carbonmonoxy hemoglobin by isotope-edited time-resolved resonance Raman spectroscopy.
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DOI：
10.1021/bi802190f
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发表时间：
2009-04-14
期刊：
影响因子：
2.9
通讯作者：
Spiro, Thomas G.
中科院分区：
文献类型：
--
作者：
Balakrishnan, Gurusamy;Zhao, Xiaojie;Podstawska, Edyta;Proniewicz, Leonard M.;Kincaid, James R.;Spiro, Thomas G.
血红蛋白是由αβ异源二聚体组成的变构四聚体蛋白。α链和β链相似，但在化学和结构上不同。为了研究链之间的动力学差异，我们已经制备了四聚体，其中链是同位素可区分的，通过重建与15 N-血红素。通过共振拉曼（RR）光谱法监测HbCO解离后配体重组和血红素结构演变的链选择性。对于α链，而不是β链，ν4卟啉呼吸模式的频率在微秒时间尺度上增加。这种增加是近端张力的Hb T状态的表现，其时间过程是平行的T接触的形成，如先前通过UVRR光谱测定。尽管α链中存在近端限制，但发现两条链中的成对重组可能性相同，产率为39 ± 2%。我们讨论了这种等价性是巧合的可能性，从某种意义上说，它来自于协同性的进化压力，或者它反映了αβ界面上的机械耦合，证据来自UVRR位点突变体的研究。
Hemoglobin is an allosteric tetrameric protein made up of αβ hetero-dimers. The α and β 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 15N-heme. Ligand recombination and heme structural evolution, following HbCO dissociation, was monitored with chain selectivity by resonance Raman (RR) spectroscopy. For α but not for β chains, the frequency of the ν4 porphyrin breathing mode increased on the microsecond time scale. This increase is a manifestation of proximal 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 α 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 αβ interface, evidence for which has emerged from UVRR studies of site-mutants.
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