TRANSIENT KINETICS OF INTRACOMPLEX ELECTRON-TRANSFER IN THE HUMAN CYTOCHROME B5 REDUCTASE-CYTOCHROME B5 SYSTEM - NAD(+) MODULATES PROTEIN-PROTEIN BINDING AND ELECTRON-TRANSFER
TRANSIENT KINETICS OF INTRACOMPLEX ELECTRON-TRANSFER IN THE HUMAN CYTOCHROME B5 REDUCTASE-CYTOCHROME B5 SYSTEM - NAD(+) MODULATES PROTEIN-PROTEIN BINDING AND ELECTRON-TRANSFER
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DOI：
10.1006/abbi.1995.1254
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发表时间：
1995-04-20
影响因子：
3.9
通讯作者：
TOLLIN, G
中科院分区：
文献类型：
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作者：
MEYER, TE;SHIRABE, K;TOLLIN, G
用激光闪光光解研究了人细胞色素b还原酶-细胞色素b(5)(b5R-b5)体系在5-去氮杂核黄素和EDTA存在下的还原和蛋白质间电子转移的瞬时动力学。脱氮杂黄素半喹酮(不含b5)闪光诱导b5R的FAD辅因子还原为快速二级反应(k(2)=3.1×10(8)M(-1)S(-1))，生成中性(蓝色)FAD半喹酮。在该体系中，细胞色素Be的血红素也迅速还原，k(2)=3.1×10(8)M(-1)，S(-1)。当这两种蛋白质在低离子强度下混合时，会形成一种很强的复合体。络合物b5的血红素能被去氮杂黄素半喹酮直接还原，但其二级速率常数比游离b5的小近一个数量级(k(2)=3.4×10(7)M(-1)S(-1))。相反，外部还原剂对b5R的FAD的降低幅度远远超过一个数量级(k(2)<；1×10(7)M(-1)S(-1))。当过量的b5R与b5的小增量滴定，然后在脱氮牛黄素/EDTA存在下进行激光闪光光解时，可以观察到蛋白质间电子从b5R FAD半喹酮转移到b5的血红素。在低离子强度(I=16 mM)下，该反应对b5浓度呈饱和行为，蛋白质间电子转移的一级极限速率常数k(1)=375 S(-1)，蛋白质-蛋白质瞬时络合物形成的解离常数约为1mM。当离子强度增加到1M时，观察到的蛋白质间电子转移速率常数下降了23倍，表明两种蛋白质之间存在正负的静电相互作用。在I=56、96和120 mm处也观察到饱和动力学，一级速率常数极限分别为195、155和63 S(-1)。在NAD(+)存在下，瞬时蛋白质-蛋白质复合体被稳定约2倍，在I=56 mM和I=96 mM时，极限一级速率常数为360 S(-1)，在I=120 mM时，极限一级速率常数为235 S(-1)。因此，NAD(+)似乎在电子转移方面稳定并优化了蛋白质-蛋白质复合体。NAD(+)的另一个作用是显著减缓FAD半喹酮的自氧化和歧化反应。细胞色素BG还增加了NAD(+)在三元络合物中的结合常数。(C)1995年学术出版社。
Transient kinetics of reduction and interprotein electron transfer in the human cytochrome bs reductase-cytochrome b(5) (b5R-b5) system was studied by laser flash photolysis in the presence of 5-deazariboflavin and EDTA at pH 7.0. Flash-induced reduction of the FAD cofactor of b5R by deazariboflavin semiquinone (in the absence of b5) occurred in a rapid second-order reaction (k(2) = 3.1 X 10(8) M(-1) s(-1)) and resulted in a neutral (blue) FAD semiquinone. The heme of cytochrome be (in the absence of b5R) was also rapidly reduced in this system with k(2) = 3.1 x 10(8) M(-1) s(-1). When the two proteins were mixed at low ionic strength, a strong complex was formed. Although the heme of complexed b5 could be directly reduced by deazariboflavin semiquinone, the second-order rate constant was nearly an order of magnitude smaller than that of free b5 (k(2) = 3.4 X 10(7) M(-1) s(-1)). In contrast, access to the FAD of b5R by the external reductant was decreased by considerably more than an order of magnitude (k(2) < 1 X 10(7) M(-1) s(-1)). When an excess of b5R was titrated with small increments of b5 and then subjected to laser flash photolysis in the presence of deazariboflavin/EDTA, interprotein electron transfer from the b5R FAD semiquinone to the heme of b5 could be observed. At low ionic strength (I = 16 mM), the reaction showed saturation behavior with respect to the b5 concentration, with a limiting first-order rate constant for interprotein electron transfer k(1) = 375 s(-1), and a dissociation constant for protein-protein transient complex formation of approximately 1 mu M. The observed rate constants for interprotein electron transfer decreased 23-fold when the ionic strength was increased to 1 M, indicating a plus-minus electrostatic interaction between the two proteins. Saturation kinetics were also observed at I = 56, 96, and 120 mM, with limiting first-order rate constants of 195, 155, and 63 s(-1), respectively. In the presence of NAD(+), the transient protein-protein complex was stabilized by approximately a factor of two, and limiting first-order rate constants of 360 s(-1) were obtained at both I = 56 mM and I = 96 mM and 235 s(-1) at I = 120 mM. Thus, NAD(+) appears to stabilize as well as to optimize the protein-protein complex with respect to electron transfer. Another effect of NAD(+) is to appreciably slow autoxidation and disproportionation of the FAD semiquinone. Cytochrome bg also increases the binding constant for NAD(+) in the ternary complex. (C) 1995 Academic Press, Inc.