Shen A L, Kasper C B
McArdle Laboratory for Cancer Research, Medical School, University of Wisconsin, Madison 53706, USA.
J Biol Chem. 1995 Nov 17;270(46):27475-80. doi: 10.1074/jbc.270.46.27475.
Site-directed mutagenesis of the acidic clusters 207Asp-Asp-Asp209 and 213Glu-Glu-Asp215 of NADPH-cytochrome P450 oxidoreductase demonstrates that both cytochrome c and cytochrome P450 interact with this region; however, the sites and mechanisms of interaction of the two substrates are clearly distinct. Substitutions in the first acidic cluster did not affect cytochrome c or ferricyanide reductase activity, but substitution of asparagine for aspartate at position 208 reduced cytochrome P450-dependent benzphetamine N-demethylase activity by 63% with no effect on KP450m or KNADPHm. Substitutions in the second acidic cluster affected cytochrome c reduction but not benzphetamine N-demethylase or ferricyanide reductase activity. The E213Q enzyme exhibited a 59% reduction in cytochrome c reductase activity and a 47% reduction in KCyt cm under standard conditions (x0.27 M potassium phosphate, pH 7.7), as well as a decreased KCyt cm at every ionic strength and a shift of the salt dependence of cytochrome c reductase activity toward lower ionic strengths. The E214Q substitution did not affect cytochrome c reductase activity under standard conditions, but shifted the salt dependence of cytochrome c reductase activity toward higher ionic strengths. Measurements of the effect of ionic strength on steady-state kinetic properties indicated that increasing ionic strength destabilized the reductase-cytochrome c3+ ground state and reductase-cytochrome c transition state complexes for the wild-type, E213Q, and E214Q enzymes, suggesting the presence of electrostatic interactions involving Glu213 and Glu214 as well as additional residues outside this region. The ionic strength dependence of kcat/KCyt cm for the wild-type and E214Q enzymes is consistent with the presence of charge-pairing interactions in the transition state and removal of a weak ionic interaction in the reductase-cytochrome c transition-state complex by the E214Q substitution. The ionic strength dependence of the E213Q enzyme, however, is not consistent with a simple electrostatic model. Effects of ionic strength on kinetic properties of E213Q suggest that substitution of glutamine stabilizes the reductase-cytochrome c3+ ground-state complex, leading to a net increase in activation energy and decrease in kcat. Glu213 is also involved in a repulsive interaction with cytochrome c3+. Cytochrome c2+ Ki for the wild-type enzyme was 82.4 microM at 118 mM ionic strength and 10.8 microM at 749 mM ionic strength; similar values were observed for the E214Q enzyme. Cytochrome c Ki for the E213Q enzyme was 17.6 microM at 118 mM and 15.7 microM at 749 mM ionic strength, consistent with removal of an electrostatic repulsion between the reductase and cytochrome c2+.
对NADPH - 细胞色素P450氧化还原酶的酸性簇207Asp - Asp - Asp209和213Glu - Glu - Asp215进行定点诱变表明，细胞色素c和细胞色素P450均与该区域相互作用；然而，两种底物的相互作用位点和机制明显不同。第一个酸性簇中的取代不影响细胞色素c或铁氰化物还原酶活性，但在第208位用天冬酰胺取代天冬氨酸可使细胞色素P450依赖性苄非他明N - 脱甲基酶活性降低63%，而对KP450m或KNADPHm无影响。第二个酸性簇中的取代影响细胞色素c的还原，但不影响苄非他明N - 脱甲基酶或铁氰化物还原酶活性。在标准条件下（0.27 M磷酸钾，pH 7.7），E213Q酶的细胞色素c还原酶活性降低59%，KCyt cm降低47%，并且在每个离子强度下KCyt cm均降低，细胞色素c还原酶活性的盐依赖性向更低离子强度偏移。E214Q取代在标准条件下不影响细胞色素c还原酶活性，但使细胞色素c还原酶活性的盐依赖性向更高离子强度偏移。离子强度对稳态动力学性质影响的测量表明，增加离子强度会使野生型、E213Q和E214Q酶的还原酶 - 细胞色素c3 +基态以及还原酶 - 细胞色素c过渡态复合物不稳定，这表明存在涉及Glu213和Glu214以及该区域外其他残基的静电相互作用。野生型和E214Q酶的kcat/KCyt cm对离子强度的依赖性与过渡态中电荷配对相互作用的存在以及E（214）Q取代消除还原酶 - 细胞色素c过渡态复合物中弱离子相互作用一致。然而，E213Q酶对离子强度的依赖性与简单的静电模型不一致。离子强度对E213Q动力学性质的影响表明，谷氨酰胺取代使还原酶 - 细胞色素c3 +基态复合物稳定，导致活化能净增加和kcat降低。Glu213还参与与细胞色素c3 +的排斥相互作用。野生型酶在118 mM离子强度下细胞色素c2 +的Ki为82.4 microM，在749 mM离子强度下为10.8 microM；E214Q酶也观察到类似值。E213Q酶在118 mM时细胞色素c的Ki为17.6 microM，在749 mM离子强度下为15.7 microM，这与还原酶和细胞色素c2 +之间静电排斥的消除一致。
