Mei H, Wang K, Peffer N, Weatherly G, Cohen D S, Miller M, Pielak G, Durham B, Millett F
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701, USA.
Biochemistry. 1999 May 25;38(21):6846-54. doi: 10.1021/bi983002t.
Electron transfer within complexes of cytochrome c (Cc) and cytochrome c peroxidase (CcP) was studied to determine whether the reactions are gated by fluctuations in configuration. Electron transfer in the physiological complex of yeast Cc (yCc) and CcP was studied using the Ru-39-Cc derivative, in which the H39C/C102T variant of yeast iso-1-cytochrome c is labeled at the single cysteine residue on the back surface with trisbipyridylruthenium(II). Laser excitation of the 1:1 Ru-39-Cc-CcP compound I complex at low ionic strength results in rapid electron transfer from RuII to heme c FeIII, followed by electron transfer from heme c FeII to the Trp-191 indolyl radical cation with a rate constant keta of 2 x 10(6) s-1 at 20 degrees C. keta is not changed by increasing the viscosity up to 40 cP with glycerol and is independent of temperature. These results suggest that this reaction is not gated by fluctuations in the configuration of the complex, but may represent the elementary electron transfer step. The value of keta is consistent with the efficient pathway for electron transfer in the crystalline yCc-CcP complex, which has a distance of 16 A between the edge of heme c and the Trp-191 indole [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. Electron transfer in the complex of horse Cc (hCc) and CcP was examined using Ru-27-Cc, in which hCc is labeled with trisbipyridylruthenium(II) at Lys-27. Laser excitation of the Ru-27-Cc-CcP complex results in electron transfer from RuII to heme c FeII with a rate constant k1 of 2.3 x 10(7) s-1, followed by oxidation of the Trp-191 indole to a radical cation by RuIII with a rate constant k3 of 7 x 10(6) s-1. The cycle is completed by electron transfer from heme c FeII to the Trp-191 radical cation with a rate constant k4 of 6.1 x 10(4) s-1. The rate constant k4 decreases to 3.4 x 10(3) s-1 as the viscosity is increased to 84 cP, but the rate constants k1 and k3 remain the same. The results are consistent with a gating mechanism in which the Ru-27-Cc-CcP complex undergoes fluctuations between a major state A with the configuration of the hCc-CcP crystalline complex and a minor state B with the configuration of the yCc-CcP complex. The hCc-CcP complex, state A, has an inefficient pathway for electron transfer from heme c to the Trp-191 indolyl radical cation with a distance of 20.5 A and a predicted value of 5 x 10(2) s-1 for k4A. The observed rate constant k4 is thus gated by the rate constant ka for conversion of state A to state B, where the rate of electron transfer k4B is expected to be 2 x 10(6) s-1. The temperature dependence of k4 provides activation parameters that are consistent with the proposed gating mechanism. These studies provide evidence that configurational gating does not control electron transfer in the physiological yCc-CcP complex, but is required in the nonphysiological hCc-CcP complex.
研究了细胞色素c（Cc）与细胞色素c过氧化物酶（CcP）复合物中的电子转移，以确定反应是否受构象波动的控制。使用Ru-39-Cc衍生物研究了酵母Cc（yCc）和CcP生理复合物中的电子转移，其中酵母同工酶-1-细胞色素c的H39C/C102T变体在背面的单个半胱氨酸残基上用三联吡啶钌（II）标记。在低离子强度下对1:1的Ru-39-Cc-CcP化合物I复合物进行激光激发，导致电子从RuII快速转移到血红素c FeIII，随后电子从血红素c FeII转移到Trp-191吲哚基自由基阳离子，在20℃时速率常数keta为2×10⁶ s⁻¹。通过加入甘油将粘度提高到40 cP，keta不变，且与温度无关。这些结果表明该反应不受复合物构象波动的控制，而可能代表基本的电子转移步骤。keta值与结晶yCc-CcP复合物中电子转移的有效途径一致，其中血红素c边缘与Trp-191吲哚之间的距离为16 Å [佩尔蒂埃，H.，和克劳特，J.（1992年）《科学》258，1748 - 1755]。使用Ru-27-Cc研究了马Cc（hCc）和CcP复合物中的电子转移，其中hCc在Lys-27处用三联吡啶钌（II）标记。对Ru-27-Cc-CcP复合物进行激光激发，导致电子从RuII转移到血红素c FeII，速率常数k1为2.3×10⁷ s⁻¹，随后RuIII将Trp-191吲哚氧化为自由基阳离子，速率常数k3为7×10⁶ s⁻¹。通过电子从血红素c FeII转移到Trp-191自由基阳离子，速率常数k4为6.1×10⁴ s⁻¹，完成循环。当粘度增加到84 cP时，速率常数k4降至3.4×10³ s⁻¹，但速率常数k1和k3保持不变。结果与一种门控机制一致，其中Ru-27-Cc-CcP复合物在具有hCc-CcP结晶复合物构象的主要状态A和具有yCc-CcP复合物构象的次要状态B之间波动。hCc-CcP复合物，即状态A，具有从血红素c到Trp-191吲哚基自由基阳离子的低效电子转移途径，距离为20.5 Å，k4A的预测值为5×10² s⁻¹。因此，观察到的速率常数k4受状态A转变为状态B的速率常数ka的控制，其中电子转移速率k4B预计为2×10⁶ s⁻¹。k4的温度依赖性提供了与所提出的门控机制一致的活化参数。这些研究提供了证据，表明构象门控在生理yCc-CcP复合物中不控制电子转移，但在非生理hCc-CcP复合物中是必需的。
