PHOTOINDUCED ELECTRON-TRANSFER BETWEEN CYTOCHROME-C PEROXIDASE AND YEAST CYTOCHROME-C LABELED AT CYS-102 WITH (4-BROMOMETHYL-4'-METHYLBIPYRIDINE)[BIS(BIPYRIDINE)]RUTHENIUM2+
PHOTOINDUCED ELECTRON-TRANSFER BETWEEN CYTOCHROME-C PEROXIDASE AND YEAST CYTOCHROME-C LABELED AT CYS-102 WITH (4-BROMOMETHYL-4'-METHYLBIPYRIDINE)[BIS(BIPYRIDINE)]RUTHENIUM2+
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DOI：
10.1021/bi00103a009
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发表时间：
1991-10-01
期刊：
影响因子：
2.9
通讯作者：
MILLETT, F
中科院分区：
文献类型：
--
作者：
GEREN, L;HAHM, S;MILLETT, F
报道了（4-溴甲基-4′-甲基联吡啶）[双（联吡啶）]六氟磷酸钌的合成。该试剂可选择性地标记酵母iso-1细胞色素c上Cys-102的单巯基，形成（二甲基联吡啶-Cys-102细胞色素c）[双（联吡啶）]钌衍生物（Ru-102-cyt c）。用短闪光激发Ru-102-cyt c形成激发态Ru(II*)，该激发态Ru（II*）迅速将一个电子转移到铁血红素基团形成Fe（II）。当细胞色素c过氧化物酶化合物I （CMPI）存在于溶液中时，观察到Ru-102-cyt c中光还原的Fe（II）向CMPI中自由基位点的电子转移。在高离子强度（100 mM磷酸钠和25 mM EDTA， pH 7）下，二级动力学观察到速率常数为（7.5 +/- 0.7）× 10(7) M-1 s-1。在此条件下，天然异-1细胞色素c的二级反应速率常数为（6.7 +/- 0.7）× 10(7) M-1 s-1。随着离子强度的降低，电子从Ru-102-cyt c转移到CMPI自由基位点的二阶速率常数增加，在40 mM EDTA， pH 7中达到（4.8 +/- 0.5）× 10(8) M-1 s-1。在较低的离子强度下，Ru-102-cyt c与CMPI形成配合物，配合物内电子向自由基位点转移的速率大于50 000 s-1。通过一系列闪光灯将CMPI滴定至CMPII，观察Ru-102-cyt c Fe（II）与CMPII中Fe（IV）位点的反应。在低离子强度（5 mM EDTA和5 mM磷酸钠，pH 7）条件下，反应速率常数为250 +/- 50 s-1，在10 mM EDTA和5 mM磷酸钠条件下，反应速率常数增加到1000 +/- 200 s-1。在更高的离子强度下，该反应变为二级反应，但在所有条件下都比CMPI中电子向自由基位点的转移慢。
The synthesis of (4-bromomethyl-4'-methylbipyridine)[bis(bipyridine)]ruthenium(II) hexafluorophosphate is described. This new reagent was found to selectively label the single sulfhydryl group at Cys-102 on yeast iso-1-cytochrome c to form the (dimethylbipyridine-Cys-102-cytochrome c)[bis(bipyridine)]ruthenium derivative (Ru-102-cyt c). Excitation of Ru-102-cyt c with a short light flash resulted in formation of excited-state Ru(II*), which rapidly transferred an electron to the ferric heme group to form Fe(II). When the cytochrome c peroxidase compound I (CMPI) was present in the solution, electron transfer from photoreduced Fe(II) in Ru-102-cyt c to the radical site in CMPI was observed. At high ionic strength (100 mM sodium phosphate and 25 mM EDTA, pH 7), second-order kinetics were observed with a rate constant of (7.5 +/- 0.7) x 10(7) M-1 s-1. The second-order rate constant for native iso-1-cytochrome c was (6.7 +/- 0.7) x 10(7) M-1 s-1 under these conditions. The second-order rate constant for electron transfer from Ru-102-cyt c to the radical site in CMPI increased as the ionic strength was decreased, reaching a value of (4.8 +/- 0.5) x 10(8) M-1 s-1 in 40 mM EDTA, pH 7. At lower ionic strength, a complex was formed between Ru-102-cyt c and CMPI, and the rate for intracomplex electron transfer to the radical site was found to be greater than 50 000 s-1. As a series of light flashes were used to titrate CMPI to CMPII, the reaction between Ru-102-cyt c Fe(II) and the Fe(IV) site in CMPII was observed. The intracomplex rate constant for this reaction was 250 +/- 50 s-1 at low ionic strength (5 mM EDTA and 5 mM sodium phosphate, pH 7) and increased to 1000 +/- 200 s-1 in 10 mM EDTA and 5 mM sodium phosphate. At still higher ionic strengths this reaction became second order, but it was slower than electron transfer to the radical site in CMPI under all conditions.