Meyer T E, Shirabe K, Yubisui T, Takeshita M, Bes M T, Cusanovich M A, Tollin G
Department of Biochemistry, University of Arizona, Tucson 85721, USA.
Arch Biochem Biophys. 1995 Apr 20;318(2):457-64. doi: 10.1006/abbi.1995.1254.
Transient kinetics of reduction and interprotein electron transfer in the human cytochrome b5 reductase-cytochrome b5 (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 (k2 = 3.1 x 10(8) M-1 s-1) and resulted in a neutral (blue) FAD semiquinone. The heme of cytochrome b5 (in the absence of b5R) was also rapidly reduced in this system with k2 = 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 (k2 = 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 (k2 < 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 k1 = 375 s-1, and a dissociation constant for protein-protein transient complex formation of approximately 1 microM. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
在pH 7.0条件下，于5 - 脱氮核黄素和乙二胺四乙酸存在的情况下，通过激光闪光光解研究了人细胞色素b5还原酶 - 细胞色素b5（b5R - b5）系统中还原及蛋白间电子转移的瞬态动力学。在无b5时，脱氮核黄素半醌使b5R的黄素腺嘌呤二核苷酸（FAD）辅因子发生闪光诱导还原，这是一个快速的二级反应（k2 = 3.1×10⁸ M⁻¹ s⁻¹），生成中性（蓝色）FAD半醌。在该系统中，细胞色素b5（无b5R时）的血红素也以k2 = 3.1×10⁸ M⁻¹ s⁻¹的速率快速还原。当两种蛋白质在低离子强度下混合时，形成了强复合物。虽然复合物b5的血红素可被脱氮核黄素半醌直接还原，但二级速率常数比游离b5的小近一个数量级（k2 = 3.4×10⁷ M⁻¹ s⁻¹）。相反，外部还原剂对b5R的FAD的作用降低幅度超过一个数量级（k2 < 1×10⁷ M⁻¹ s⁻¹）。当用少量b5逐步滴定过量的b5R，然后在脱氮核黄素/乙二胺四乙酸存在下进行激光闪光光解时，可观察到从b5R FAD半醌到b5血红素的蛋白间电子转移。在低离子强度（I = 16 mM）下，该反应对b5浓度呈现饱和行为，蛋白间电子转移的极限一级速率常数k1 = 375 s⁻¹，蛋白质 - 蛋白质瞬态复合物形成的解离常数约为1 μM。当离子强度增加到1 M时，观察到的蛋白间电子转移速率常数降低了23倍，表明两种蛋白质之间存在正负静电相互作用。在I = 56、96和120 mM时也观察到饱和动力学，极限一级速率常数分别为195、155和63 s⁻¹。在烟酰胺腺嘌呤二核苷酸（NAD⁺）存在下时，瞬态蛋白质 - 蛋白质复合物稳定了约两倍，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时为235 s⁻¹。因此，NAD⁺似乎既能稳定蛋白质 - 蛋白质复合物，又能优化其电子转移。NAD⁺的另一个作用是显著减缓FAD半醌的自动氧化和歧化反应。（摘要截断于250字）
