Pappa H S, Tajbaksh S, Saunders A J, Pielak G J, Poulos T L
Department of Molecular Biology, University of California at Irvine, 92717, USA.
Biochemistry. 1996 Apr 16;35(15):4837-45. doi: 10.1021/bi952935b.
Engineered cysteine residues in yeast cytochrome c peroxidase (CCP) and yeast iso-1-cytochrome c have been used to generate site specifically cross-linked peroxidase-cytochrome c complexes for the purpose of probing interaction domains and the intramolecular electron transfer reaction. Complex 2 was designed earlier [Pappa, H.S., & Poulos, T.L. (1995) Biochemistry 34, 6573-6580] to mimic the known crystal structure of the peroxidase-cytochrome c noncovalent complex [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. Complex 3 was designed such that cytochrome c is tethered to a region of the peroxidase near Asp148 which has been suggested to be a second site of interaction between the peroxidase and cytochrome c. Using stopped flow methods, the rate at which the ferrocytochrome c covalently attached to the peroxidase transfers an electron to peroxidase compound I is estimated to be approximately 0.5-1 s-1 in complex 3 and approximately 800 s-1 in complex 2. In both complexes the Trp191 radical and not the Fe4+=O oxyferryl center of compound I is reduced. Conversion of Trp191 to Phe slows electron transfer about 10(3) in complex 2. Steady state kinetic measurements show that complex 3 behaves like the wild type enzyme when either horse heart or yeast ferrocytochrome c is used as an exogenous substrate, indicating that the region blocked in complex 3 is not a functionally important interaction site. In contrast, complex 2 is inactive toward horse heart ferrocytochrome c at all ionic strengths tested and yeast ferrocytochrome c at high ionic strengths. Only at low ionic strengths and low concentrations of yeast ferrocytochrome c does complex 2 give wild type enzyme activity. This observation indicates that in complex 2 the primary site of interaction of CCP with horse heart and yeast ferrocytochrome c at high ionic strengths is blocked. The relevance of these results to the pathway versus distance models of electron transfer and to the interaction domains between peroxidase and cytochrome c is discussed.
酵母细胞色素c过氧化物酶（CCP）和酵母同工-1-细胞色素c中经工程改造的半胱氨酸残基已被用于生成位点特异性交联的过氧化物酶-细胞色素c复合物，以探究相互作用结构域和分子内电子转移反应。复合物2是较早设计的[Pappa, H.S., & Poulos, T.L. (1995) Biochemistry 34, 6573 - 6580]，用于模拟过氧化物酶-细胞色素c非共价复合物的已知晶体结构[Pelletier, H., & Kraut, J. (1992) Science 258, 1748 - 1755]。复合物3的设计使得细胞色素c与过氧化物酶靠近Asp148的区域相连，该区域被认为是过氧化物酶与细胞色素c之间的第二个相互作用位点。使用停流方法，估计在复合物3中与过氧化物酶共价连接的亚铁细胞色素c将电子转移到过氧化物酶化合物I的速率约为0.5 - 1 s-1，在复合物2中约为800 s-1。在这两种复合物中，被还原的是Trp191自由基而非化合物I的Fe4+=O氧合铁中心。在复合物2中，将Trp191替换为Phe会使电子转移减慢约10³倍。稳态动力学测量表明，当使用马心或酵母亚铁细胞色素c作为外源底物时，复合物3的行为类似于野生型酶，这表明复合物3中被阻断的区域不是功能上重要的相互作用位点。相比之下，复合物2在所有测试的离子强度下对马心亚铁细胞色素c均无活性，在高离子强度下对酵母亚铁细胞色素c也无活性。只有在低离子强度和低浓度的酵母亚铁细胞色素c条件下，复合物2才具有野生型酶活性。这一观察结果表明，在复合物
