Alkaline Conformational Transitions of FerricytochromecStudied by Resonance Raman Spectroscopy

作者：Susanne Döpner, Peter Hildebrandt, Federico I. Rosell, A. Grant Mauk · 发表于：Journal of the American Chemical Society · 年份：1998 · DOI：10.1021/ja9717572 · 被引用次数：142 · 研究领域：Hemoglobin structure and function、Protein Interaction Studies and Fluorescence Analysis、Photosynthetic Processes and Mechanisms

The pH-dependent conformational equilibria of iso-1-ferricytochrome c that occur between pH 7 and pH 12 have been studied by resonance Raman (RR) spectroscopy. Detailed analysis of the RR spectra provides the number and the spectra of the conformational species that occur over this range of pH as well as their relative concentrations at each pH. Between pH 7 and pH 12, the wild-type protein exhibits five conformers which differ with respect to the axial ligands of the heme iron. In the medium alkaline pH range, the neutral form undergoes two parallel conformational transitions with similar p K a s (8.7 and 8.9) to those of the states IVa and IVb. Comparative studies with protein variants in which either Lys79 or Lys73 or both lysyl residues were replaced by alanine(s) confirm previous conclusions (Rosell et al., preceding paper in this issue) that Lys73 and Lys79 are the axial ligands replacing Met80 in the states IVa and IVb, respectively. On the basis of isotopic shifts caused by H/D exchange, the Fe−N(Lys) stretching vibrations of these species were identified at 385 cm - 1 . In the high alkaline pH range, the wild-type protein forms two further conformers with p K a s of 10.5 and 11. In these states, Va and Vb, which are also detected in the Lys73Ala, Lys79Ala, and the Lys73Ala/Lys79Ala variant proteins, another strong-field ligand, presumably a hydroxide, occupies the sixth axial coordination site. Comparison of the RR spectra demonstrates far-reaching similarities betwe...