Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, 41125, Modena, Italy.
J Biol Inorg Chem. 2011 Mar;16(3):461-71. doi: 10.1007/s00775-010-0742-y. Epub 2010 Dec 16.
The 16-kDa diheme cytochrome c from the bacterium Shewanella baltica OS155 (Sb-DHC) was cloned and expressed in Escherichia coli and investigated through UV-vis, magnetic circular dichroism, and (1)H NMR spectroscopies and protein voltammetry. The model structure was obtained by means of comparative modeling using the X-ray structure of Rhodobacter sphaeroides diheme cytochrome c (Rs-DHC) (with a 37% pairwise sequence identity) as a template. Sb-DHC folds into two distinct domains, each containing one heme center with a bis-His axial ligation. Both secondary and tertiary structures of the N-terminal domain resemble those of class I cytochrome c, displaying three α-helices and a compact overall folding. The C-terminal domain is less helical than the corresponding domain of Rs-DHC. The two heme groups are bridged by Tyr26 in correspondence with the shortest edge-to-edge distance, a feature which would facilitate fast internal electron transfer. The electronic properties of the two prosthetic centers are equivalent and sensitive to two acid-base equilibria with pK (a) values of approximately 2.4 and 5, likely corresponding to protonation and detachment of the axial His ligands from the heme iron and a pH-linked conformational change of the protein, respectively. Reduction potentials of -0.144 and -0.257 V (vs. the standard hydrogen electrode), were determined for the C- and N-terminal heme groups, respectively. An approach based on the extended Debye-Hückel equation was applied for the first time to a two-centered metalloprotein and was found to reproduce successfully the ionic strength dependence of E°'.
从细菌希瓦氏菌 OS155（Sb-DHC）中克隆并表达了 16kDa 二血红素细胞色素 c，并通过紫外可见光谱、圆二色性和（1）H NMR 光谱和蛋白伏安法进行了研究。通过使用 X 射线结构的比较建模获得了模型结构罗尔斯顿氏菌二血红素细胞色素 c（Rs-DHC）（具有 37％的序列同一性）作为模板。Sb-DHC 折叠成两个不同的结构域，每个结构域包含一个双组氨酸轴向配位的血红素中心。N 端结构域的二级和三级结构类似于 I 类细胞色素 c，显示出三个α-螺旋和紧凑的整体折叠。C 端结构域比 Rs-DHC 的相应结构域少螺旋。两个血红素基团通过 Tyr26 桥接，对应于最短的边缘到边缘距离，这一特征将促进快速的内部电子转移。两个辅基中心的电子性质是等效的，并且对两个酸碱平衡敏感，pK（a）值约为 2.4 和 5，可能对应于轴向 His 配体从血红素铁上的质子化和脱附以及蛋白质的 pH 相关构象变化。分别确定了 C-和 N-末端血红素中心的还原电位为-0.144 和-0.257 V（相对于标准氢电极）。基于扩展的德拜-休克尔方程的方法首次应用于双中心金属蛋白，并成功地再现了 E°'的离子强度依赖性。
