Modulation of redox potential in electron transfer proteins:: Effects of complex formation on the active site microenvironment of cytochrome b5
Modulation of redox potential in electron transfer proteins:: Effects of complex formation on the active site microenvironment of cytochrome b5
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DOI：
10.1039/b001520m
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发表时间：
2000-01-01
影响因子：
3.4
通讯作者：
Rivera, M
中科院分区：
文献类型：
--
作者：
Wirtz, M;Oganesyan, V;Rivera, M
细胞色素B（5）的还原电位通过在电极表面与多聚赖氨酸形成复合物来调节（里维拉等人，Biochemistry，1998，37，1485）。这种调制被认为是源于溶剂暴露的血红素丙酸盐的中和和复杂界面的脱水。虽然已经获得了证明血红素丙酸酯上电荷的中和有助于调节细胞色素B（5）氧化还原电位的直接证据，但证明络合物界面的水排斥作用起类似作用的证据尚未得出结论。本文报道了大鼠肝线粒体外膜（OM）细胞色素B的V45 I/V61 I双突变体的制备（5）。该突变体的设计目的是限制水对细胞色素B暴露的血红素边缘的可及性（5）。V45 I/V61 I突变体的X射线晶体结构显示，Ile在位置45和61的侧链限制了血红素腔内部的水可及性，并保护了血红素边缘的大部分免受水性环境的影响。用细胞色素B的V45 I/V61 I突变体（5）和其中血红素丙酸酯已转化为相应的二甲酯基团的衍生物进行的电化学研究清楚地表明，血红素边缘的脱水有助于调节细胞色素B的还原电位（5）。事实上，这些研究表明，从复合物界面排除水产生的效果（类似于40 mV偏移）与源自溶剂暴露的血红素丙酸酯上的电荷中和的效果（类似于30 mV偏移）相当，如果不是更大的话。
The reduction potential of cytochrome b(5) is modulated via the formation of a complex with polylysine at the electrode surface (Rivera et al., Biochemistry, 1998, 37, 1485). This modulation is thought to originate from the neutralization of a solvent exposed heme propionate and from dehydration of the complex interface. Although direct evidence demonstrating that neutralization of the charge on the heme propionate contributes to the modulation of the redox potential of cytochrome b(5) has been obtained, evidence demonstrating that water exclusion from the complex interface plays a similar role has not been conclusive. Herein we report the preparation of the V45I/V61I double mutant of rat liver outer mitochondrial membrane (OM) cytochrome b(5). This mutant has been engineered with the aim of restricting water accessibility to the exposed heme edge of cytochrome b(5). The X-ray crystal structure of the V45I/V61I mutant revealed that the side chain of Ile at positions 45 and 61 restricts water accessibility to the interior of the heme cavity and protects a large section of the heme edge from the aqueous environment. Electrochemical studies performed with the V45I/V61I mutant of cytochrome b(5), and with a derivative in which the heme propionates have been converted into the corresponding dimethyl ester groups, clearly demonstrate that dehydration of the heme edge contributes to the modulation of the reduction potential of cytochrome b(5). In fact, these studies showed that exclusion of water from the complex interface exerts an effect (similar to 40 mV shift) that is comparable, if not larger, than the one originating from neutralization of the charge on the solvent exposed heme propionate (similar to 30 mV shift).