Dumortier C, Holt J M, Meyer T E, Cusanovich M A
Department of Biochemistry, University of Arizona, Tucson, Arizona 85721, USA.
J Biol Chem. 1998 Oct 2;273(40):25647-53. doi: 10.1074/jbc.273.40.25647.
Although ligand binding in c-type cytochromes is not directly related to their physiological function, it has the potential to provide valuable information on protein stability and dynamics, particularly in the region of the methionine sixth heme ligand and the nearby peptide chain that has been implicated in electron transfer. Thus, we have measured the equilibrium and kinetics of binding of imidazole to eight mutants of Rhodobacter capsulatus cytochrome c2 that differ in overall protein stability. We found that imidazole binding affinity varies 70-fold, but does not correlate with overall protein stability. Instead, each mutant exerts an effect at the local level, with the largest change due to mutant G95E (glycine substituted by glutamate), which shows 30-fold stronger binding as compared with the wild-type protein. The kinetics of imidazole binding are monophasic and reach saturation at high ligand concentrations for all the mutants and wild-type protein, which is attributed to a rate-limiting conformational change leading to breakage of the iron-methionine bond and providing a binding site for imidazole. The mutants show as much as an 18-fold variation in the first-order rate constant for the conformational change, with the largest effect found with mutant G95E. The kinetics also show a lack of correlation with overall protein stability, but are consistent with localized effects on the dynamics of hinge region 88-102 of the protein, which changes conformation to permit ligand binding. These results are consistent with R. capsulatus cytochrome c2 stabilizing the complex through hydrogen bonding to the imidazole. The larger effects of mutant G95E on equilibrium and kinetics are likely to be due to its location within the hinge region adjacent to heme ligand methionine 96, which is displaced by imidazole.
尽管c型细胞色素中的配体结合与其生理功能没有直接关系，但它有可能提供有关蛋白质稳定性和动力学的有价值信息，特别是在甲硫氨酸第六个血红素配体区域以及与电子转移有关的附近肽链区域。因此，我们测量了咪唑与八株荚膜红细菌细胞色素c2突变体的结合平衡和动力学，这些突变体在整体蛋白质稳定性上有所不同。我们发现咪唑结合亲和力变化了70倍，但与整体蛋白质稳定性无关。相反，每个突变体在局部水平上产生影响，其中突变体G95E（甘氨酸被谷氨酸取代）引起的变化最大，与野生型蛋白质相比，其结合力强30倍。对于所有突变体和野生型蛋白质，咪唑结合动力学是单相的，并且在高配体浓度下达到饱和，这归因于限速构象变化导致铁 - 甲硫氨酸键断裂并为咪唑提供结合位点。突变体在构象变化的一级速率常数上表现出高达18倍的变化，其中突变体G95E的影响最大。动力学也显示与整体蛋白质稳定性缺乏相关性，但与对蛋白质铰链区88 - 102动力学的局部影响一致，该区域改变构象以允许配体结合。这些结果与荚膜红细菌细胞色素c2通过与咪唑形成氢键来稳定复合物一致。突变体G95E对平衡和动力学的较大影响可能是由于其位于与血红素配体甲硫氨酸96相邻的铰链区内，该区域被咪唑取代。
