Zhao D, Hutton H M, Gooley P R, MacKenzie N E, Cusanovich M A
Department of Pharmacology & Toxicology, College of Pharmacy, University of Arizona, Tucson 85721, USA.
Protein Sci. 2000 Sep;9(9):1828-37. doi: 10.1110/ps.9.9.1828.
WEFT-NOESY and transfer WEFT-NOESY NMR spectra were used to determine the heme proton assignments for Rhodobacter capsulatus ferricytochrome c2. The Fermi contact and pseudo-contact contributions to the paramagnetic effect of the unpaired electron in the oxidized state were evaluated for the heme and ligand protons. The chemical shift assignments for the 1H and 15N NMR spectra were obtained by a combination of 1H-1H and 1H-15N two-dimensional NMR spectroscopy. The short-range nuclear Overhauser effect (NOE) data are consistent with the view that the secondary structure for the oxidized state of this protein closely approximates that of the reduced form, but with redox-related conformational changes between the two redox states. To understand the decrease in stability of the oxidized state of this cytochrome c2 compared to the reduced form, the structural difference between the two redox states were analyzed by the differences in the NOE intensities, pseudo-contact shifts and the hydrogen-deuterium exchange rates of the amide protons. We find that the major difference between redox states, although subtle, involve heme protein interactions, orientation of the heme ligands, differences in hydrogen bond networks and, possible alterations in the position of some internal water molecules. Thus, it appears that the general destabilization of cytochrome c2, which occurs on oxidation, is consistent with the alteration of hydrogen bonds that result in changes in the internal dynamics of the protein.
利用WEFT - NOESY和转移WEFT - NOESY核磁共振谱来确定荚膜红细菌铁细胞色素c2的血红素质子归属。对血红素和配体质子评估了氧化态下未成对电子顺磁效应的费米接触和赝接触贡献。通过1H - 1H和1H - 15N二维核磁共振光谱相结合的方法获得了1H和15N核磁共振谱的化学位移归属。短程核Overhauser效应（NOE）数据与以下观点一致：该蛋白质氧化态的二级结构与还原态的二级结构非常相似，但两种氧化还原状态之间存在与氧化还原相关的构象变化。为了理解这种细胞色素c2氧化态与还原态相比稳定性降低的原因，通过NOE强度、赝接触位移和酰胺质子的氢 - 氘交换速率的差异分析了两种氧化还原状态之间的结构差异。我们发现，氧化还原状态之间的主要差异虽然细微，但涉及血红素与蛋白质的相互作用、血红素配体的取向、氢键网络的差异以及一些内部水分子位置可能的改变。因此，似乎细胞色素c2在氧化时发生的总体不稳定与氢键的改变有关，而氢键的改变导致了蛋白质内部动力学的变化。
