Role of Configurational Gating in Intracomplex Electron Transfer from Cytochrome c to the Radical Cation in Cytochrome c Peroxidase

Abstract

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.

Metalloprotein Association, Self-Association, and Dynamics Governed by Hydrophobic Interactions: Simultaneous Occurrence of Gated and True Electron-Transfer Reactions between Cytochrome f and Cytochrome c6 from Chlamydomonas reinhardtii

“…Two simultaneous first-order reactions are detected in each case, but more than two complexes may perhaps be present. 45,46,51,[78][79][80][81][82][83] The variety of kinetic results in these three studies shows the diversity of dynamic properties of fairly similar donor-acceptor systems, all of which are mostly held by electrostatic forces. No two of these systems show the same effects and noneffects of ionic strength and viscosity on the intracomplex rate constant.…”

Effects of interface mutations on association modes and electron-transfer rates between proteins

“…Reaction rates depend on factors that control both electron tunneling and conformational dynamics coupled to protein association processes (1)(2)(3). As such, interprotein ET is sensitive to structure and dynamics at the interface (1,(4)(5)(6)(7)(8)(9)(10)(11). Residue substitution (achieved either by use of protein homologs, site-directed mutants, or computations) has been a popular and powerful approach for probing how interface composition influences interprotein ET (7,(10)(11)(12)(13)(14).…”

Electron tunneling in protein crystals

“…Accordingly, we have measured and analyzed the kinetics of electron transfer between native and Zn-substituted tuna cytochrome c (cyt c) molecules in crystals of known structure. E xtensive experimental and theoretical investigations have elucidated the role of polypeptide structure in facilitating electron tunneling through proteins (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Most of the definitive work has centered on molecules with fixed donor-acceptor distances and orientations, such as proteins covalently modified with redox-active units (1)(2)(3)(4)(5) or proteins that contain both donors and acceptors (6,7).…”

“…E xtensive experimental and theoretical investigations have elucidated the role of polypeptide structure in facilitating electron tunneling through proteins (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Most of the definitive work has centered on molecules with fixed donor-acceptor distances and orientations, such as proteins covalently modified with redox-active units (1)(2)(3)(4)(5) or proteins that contain both donors and acceptors (6,7). This work has established that the dependence of rate on distance is exponential (1,2,12,13), as expected for a tunneling reaction (8), with decay constants in the 1.0-to 1.2-Å Ϫ1 range (1,2,13).…”

“…Because the dynamics of the first and the third steps obscure the electron tunneling reaction, many studies have focused on the ET properties of stable protein-protein complexes in solution (5,15,17). It has been difficult to interpret the results, however, as neither the donor-acceptor docking geometries nor the conformations of these complexes are known.…”