Liu R Q, Miller M A, Han G W, Hahm S, Geren L, Hibdon S, Kraut J, Durham B, Millett F
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701.
Biochemistry. 1994 Jul 26;33(29):8678-85. doi: 10.1021/bi00195a008.
The kinetics of electron transfer from cytochrome c (CC) to yeast cytochrome c peroxidase (CcP) compound I were studied by flash photolysis and stopped-flow spectroscopy. Flash photolysis studies employed horse CC derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)bis-(bipyridine)ruthenium (Ru-CC). Initial electron transfer from Ru-CC reduced the indole radical on Trp-191 of CcP compound I [CMPI(IV,R.)], producing CMPII(IV,R). This reaction was biphasic for each of several Ru-CC derivatives, with rate constants which varied according to the position of the Ru label. For Ru-27-CC labeled at lysine 27, rate constants of 43,000 and 1600 s-1 were observed at pH 5.0 in 2 mM acetate. After reduction of the indole radical by Ru-CC, intramolecular electron transfer from Trp-191 to the oxyferryl heme in CMPII(IV,R) was observed, producing CMPII(III,R.). The rate constant and extent of this intramolecular electron transfer reaction were independent of both the protein concentration and the Ru-CC derivative employed. The rate constant decreased from 1100 s-1 at pH 5 to 550 s-1 at pH 6, while the extent of conversion of CMPII(IV,R) to CMPII(III,R.) decreased from 56% at pH 5 to 29% at pH 6. The reaction was not detected at pH 7.0 and above. The pH dependence of the rate and extent of this internal electron transfer reaction paralleled the pH dependence of the rate of bimolecular reduction of CMPII(IV,R) by native horse CC measured by stopped-flow spectroscopy at high ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)
通过闪光光解和停流光谱法研究了电子从细胞色素c（CC）转移至酵母细胞色素c过氧化物酶（CcP）化合物I的动力学。闪光光解研究使用了在特定赖氨酸氨基上标记有（二羧基联吡啶）双（联吡啶）钌（Ru-CC）的马CC衍生物。来自Ru-CC的初始电子转移还原了CcP化合物I [CMPI(IV,R.)]中色氨酸-191上的吲哚自由基，生成CMPII(IV,R)。对于几种Ru-CC衍生物中的每一种，该反应都是双相的，速率常数根据Ru标记的位置而变化。对于在赖氨酸27处标记的Ru-27-CC，在pH 5.0的2 mM乙酸盐中观察到的速率常数分别为43,000和1600 s-1。在Ru-CC还原吲哚自由基后，观察到了从色氨酸-191到CMPII(IV,R)中氧合铁血红素的分子内电子转移，生成CMPII(III,R.)。该分子内电子转移反应的速率常数和程度与蛋白质浓度以及所使用的Ru-CC衍生物均无关。速率常数从pH 5时的1100 s-1降至pH 6时的550 s-1，而CMPII(IV,R)转化为CMPII(III,R.)的程度从pH 5时的56%降至pH 6时的29%。在pH 7.0及以上未检测到该反应。这种内部电子转移反应的速率和程度对pH的依赖性与在高离子强度下通过停流光谱法测量的天然马CC对CMPII(IV,R)的双分子还原速率对pH的依赖性相似。（摘要截断于250字）
