Yu N T, Mackin Thompson H, Zepke D, Gersonde K
Eur J Biochem. 1986 Jun 16;157(3):579-83. doi: 10.1111/j.1432-1033.1986.tb09705.x.
Resonance Raman spectroscopy has been used to investigate the allosteric control mechanism for O2 binding in a cobalt-substituted dimeric insect hemoglobin (CTT II), which exhibits a large Bohr effect due to a pH-induced transition between two ligand affinity states. Substitution of cobalt for iron in CTT II does not modify the Bohr effect, but permits the resonance enhancement (hence the detection) of Raman lines corresponding to the vibrations of the axial ligand-cobalt bonds. Using 16O2/18O2 isotope substitution the O-O and Co-O2 stretching and the Co-O-O bending mode have been assigned to the two affinity states of this hemoglobin: v (O-O) changes from 1152 cm-1 (pH 5.5; t conformation) to about 1125 cm-1 (pH 9.5, r conformation), v (Co-O2) from 512 cm-1 (pH 5.5) to 537 cm-1 (pH 9.5) and delta (Co-O-O) from 378 cm-1 (pH 5.5) to 390 cm-1 (pH 9.5). The Co-N epsilon (His) stretching mode has also been detected changing from 313 cm-1 (pH 5.5) to 307 cm-1 (pH 9.5). For the first time, reciprocal behaviour between the Co-N epsilon and Co-O2 bonds and between the Co-O2 and the O-O bonds in an allosteric hemoglobin are demonstrated. Furthermore, the pH sensitivity of a vinyl bending mode in the range of 411-415 cm-1 has been investigated and shown also to reflect the t in equilibrium with r conformation transition.
共振拉曼光谱已被用于研究钴取代的二聚体昆虫血红蛋白（CTT II）中O₂结合的变构控制机制，该血红蛋白由于在两种配体亲和状态之间发生pH诱导的转变而表现出较大的玻尔效应。用钴取代CTT II中的铁不会改变玻尔效应，但能使与轴向配体 - 钴键振动相对应的拉曼谱线产生共振增强（从而实现检测）。使用¹⁶O₂/¹⁸O₂同位素取代，已将O - O和Co - O₂伸缩振动以及Co - O - O弯曲模式归属于该血红蛋白的两种亲和状态：v（O - O）从1152 cm⁻¹（pH 5.5；t构象）变为约1125 cm⁻¹（pH 9.5，r构象），v（Co - O₂）从512 cm⁻¹（pH 5.5）变为537 cm⁻¹（pH 9.5），δ（Co - O - O）从378 cm⁻¹（pH 5.5）变为390 cm⁻¹（pH 9.5）。还检测到Co - Nε（His）伸缩模式从313 cm⁻¹（pH 5.5）变为307 cm⁻¹（pH 9.5）。首次证明了变构血红蛋白中Co - Nε与Co - O₂键之间以及Co - O₂与O - O键之间的相互作用。此外，还研究了411 - 415 cm⁻¹范围内乙烯基弯曲模式的pH敏感性，结果表明它也反映了t构象与r构象转变的平衡。
