Kinetics of intracomplex electron transfer and of reduction of the components of covalent and noncovalent complexes of cytochrome c and cytochrome c peroxidase by free flavin semiquinones.
Kinetics of intracomplex electron transfer and of reduction of the components of covalent and noncovalent complexes of cytochrome c and cytochrome c peroxidase by free flavin semiquinones.
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复合物内电子转移以及游离黄素半醌还原细胞色素 c 和细胞色素 c 过氧化物酶的共价和非共价复合物成分的动力学。
DOI：
10.1021/bi00406a029
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Tollin,G
中科院分区：
文献类型：
--
作者：
Hazzard,JT;Moench,SJ;Erman,JE;Satterlee,JD;Tollin,G
摘要：研究了酵母铁和铁基细胞色素c过氧化物酶和铁马细胞色素c的1：1共价和静电复合物的单个组分的游离黄素半醌的还原动力学。过氧化物酶和细胞色素c在低离子强度下的共价交联导致复合物具有与静电复合物相似和不同的动力学性质。而细胞色素c血红素暴露于外源性还原剂是相似的两个复合物，表观静电环境附近的细胞色素c血红素边缘是显着不同的。在静电复合物中，存在一个净正电荷，而在共价复合物中，存在一个基本上中性的静电电荷。对于共价复合物，从亚铁细胞色素c到铁基过氧化物酶的电子转移具有1560 s-1的速率常数，其相对于离子强度的变化是不变的。在静电复合物内的复合物内电子转移的速率常数是高度依赖于离子强度。在μ= 8 mM时，已获得750 s-1的值[Hazzard，J.T.，Poulos，T. L.，& Tollin，G.（1987）Biochemistry 26，2836-2848]，而在μ= 30 mM时，该值为3300 s-1。静电复合物的这种离子强度依赖性已被解释为两种蛋白质的重排，包括复合物的电子转移到一个更有利的方向。在共价复合物的情况下，这种重新取向显然受到阻碍。这些动力学结果根据Poulos和Kraut [Poulos，T. J，& J.（1980）J.Biol.Chem.255，10322-10330]和Waldmeyer和Bosshard的建议[Waldmeyer，B.，& Bosshard，H. R.（1985）J.Biol.Chem.260，5184-5190]关于共价交联位点的位置。人们对静电稳定的1：1氧化还原蛋白复合物的结构性质表现出极大的兴趣，以便更充分地理解控制复合物内电子转移动力学的因素。人们普遍认为，吸引性静电相互作用负责将两种蛋白质以这样的方式结合在一起，使得辅基具有有利的距离和方向，以便电子从一种蛋白质转移到另一种蛋白质（Salemme，1978）。已经基于计算机图形建模提出了电子转移络合物的几种假设结构（Salemme，1976; Simondsen等人，1982年; Poulos和Kraut，1980年; Poulos和莫克，1983年;莫克等人，1986年）。在所有这些情况下，互补的静电相互作用，优化氧化还原中心之间的相互取向和距离一直是在建模过程中的一个主要标准。如果静电力确实是使反应伙伴最佳定向的主要因素，可以合理地预期，与电子转移有关的络合物缔合常数和络合物内过程的一级速率常数这项工作得到了NIH对GT（AM 15057）和NIH（HL 01758）的资助。和NSF（DMB 8403353）以及Alfred P. Sloan基金会向JDS提供的奖学金，这些研究的初步结果由Hazzard et al.（1987年b）。
Revised Manuscript Received November 16, 1987 abstract: The kinetics of reduction of free flavin semiquinones of the individual components of 1: 1 covalent and electrostatic complexes of yeast ferric and ferryl cytochrome c peroxidase and ferric horse cytochrome c have been studied. Covalent cross-linking between the peroxidase and cytochrome c at low ionic strength results in a complex that has kinetic properties both similar to and different from those of the electrostatic complex. Whereas the cytochrome c heme exposure to exogenous reductants is similar in bothcomplexes, the apparent electrostatic environment near the cytochrome c heme edge is markedly different. In the electrostatic complex, a net positive charge is present, whereas in the covalent complex, an essentially neutral electrostatic charge is found. Intracomplex electron transfer within the two complexes is also different. For the covalent complex, electron transfer from ferrous cytochrome c to the ferryl peroxidase has a rate constant of 1560 s'1, which is invariant with respect to changes in the ionic strength. The rate constant for intracomplex electron transfer within the electrostatic complex is highly ionic strength dependent. At µ= 8 mM a value of 750 s'1 has been obtained [Hazzard, J. T., Poulos, T. L., & Tollin, G.(1987) Biochemistry 26, 2836-2848], whereas at µ= 30 mM the value is 3300 s'1. This ionic strength dependency for the electrostatic complex has been interpreted in terms of the rearrangement of the two proteins comprising the complex to a more favorable orientation for electron transfer. In the case of the covalent complex, such reorientation is apparently impeded. These kinetic results are discussed in terms of the hypothetical model for the complex proposed by Poulos and Kraut [Poulos, T. L, & Kraut, J.(1980) J. Biol. Chem. 255, 10322-10330] and the proposal of Waldmeyer and Bosshard [Waldmeyer, B., & Bosshard, H. R.(1985) J. Biol. Chem. 260, 5184-5190] regarding the position of sites of covalent cross-linking. great deal of interest has been shown in the structural nature of electrostatically stabilized 1: 1 redox protein com-plexes in order to more fully understand the factors that govern intracomplex electron transfer kinetics. It has generally been accepted that attractive electrostatic interactions are responsible for bringing the two proteins together in such a manner that the prosthetic groups have a favorable distance and orientation for electron transfer from one protein to the other (Salemme, 1978). Several hypothetical structures for elec-tron-transfer complexes have been proposed on the basis of computer graphics modeling (Salemme, 1976; Simondsen et al., 1982; Poulos & Kraut, 1980; Poulos & Mauk, 1983; Mauk et al., 1986). In all these cases, complementary electrostatic interactions which optimize the mutual orientation and distance between redox centers have been a predominant criterion in the model building procedure. If electrostatic forces are indeed a primary factor in optimally orienting the reaction partners, it is reasonable to expect that both the complex association constant and the first-order rate constant for the intracomplex processes associated with electron transfer (these would include any reorientations that influence redoxcenter• This work was supported by a grant from NIH to GT (AM 15057) and by grantsfrom NIH (HL01758) and NSF (DMB8403353) and a fellowship from the Alfred P. Sloan Foundation to JDS Preliminary results from these studies were presented in abstractform by Hazzard et al.(1987b).