Intersubunit Communication via Changes in Hemoglobin Quaternary Structures Revealed by Time-Resolved Resonance Raman Spectroscopy: Direct Observation of the Perutz Mechanism

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• 作者：Kenta Yamada ; Haruto Ishikawa ; Misao Mizuno ; Naoya Shibayama ; Yasuhisa Mizutani
• 刊名：Journal of Physical Chemistry B
• 出版年：2013
• 出版时间：October 17, 2013
• 年：2013
• 卷：117
• 期：41
• 页码：12461-12468
• 全文大小：411K
• 年卷期：v.117,no.41(October 17, 2013)
• ISSN：1520-5207

文摘

Time-resolved resonance Raman spectroscopy was used to investigate intersubunit communication of hemoglobin using hybrid hemoglobin in which nickel was substituted for the heme iron in the 尾 subunits. Changes in the resonance Raman spectra of the 伪 heme and the 尾 Ni鈥揾eme groups in the hybrid hemoglobin were observed upon CO photolysis in the 伪 subunit using a probe pulse of 436 and 418 nm, respectively. Temporal evolution of the frequencies of the 谓(Fe鈥揌is) and the 纬₇ band of 伪 heme was similar to that of unsubstituted hemoglobin, suggesting that substitution with Ni鈥揾eme did not perturb the allosteric dynamics of the hemoglobin. In the 尾 subunits, no structural change in the Ni鈥揾eme was observed until 1 渭s. In the microsecond regime, temporal evolution of the frequencies of the 谓(Ni鈥揌is) and the 纬₇ band of 尾 Ni鈥揾eme was observed concomitant with an R 鈫?T quaternary change at about 20 渭s. The changes in the 谓(Fe鈥揌is) and 谓(Ni鈥揌is) frequencies of the 伪 and 尾 subunits with the common time constant of 20 渭s indicated that the proximal tension imposed on the bond between the heme and the proximal histidine strengthened upon the quaternary changes in both the 伪 and the 尾 subunits concertedly. This observation is consistent with the Perutz mechanism for allosteric control of oxygen binding in hemoglobin and, thus, is the first real-time observation of the mechanism. Protein dynamics and allostery based on the observed time-resolved spectra also are discussed.