Investigation of the Electron-Transfer Mechanism by Cross-Linking between Zn-substituted Myoglobin and Cytochromeb5

Publisher Website
Google Scholar
Abstract
We have investigated the photoinduced electron transfer (ET) in the 1:1 cross-linked complex (CL-ZnMb/b5) formed by a cross-linking reagent, EDC, between Zn-substituted myoglobin (ZnMb) and cytochrome b5 (Cytb5) to reveal the mechanism of the inter-protein ET reactions under the condition of multiple encounter complexes. A variety of the ZnMb−Cytb5 orientations was suggested because of failure to identify the single and specific cross-linking site on Cytb5 by the peptide-mapping analysis using mass spectrometry. In CL-ZnMb/b5, a laser pulse generates the triplet excited state of the ZnMb domain (3ZnMb*), which can transfer one electron to the Cytb5 domain. The decay kinetics of 3ZnMb* in CL-ZnMb/b5 consists of a facile power-law ET phase to Cytb5 domain (∼30%) and a slower single-exponential phase (∼70%). The application of the Marcus equation to this power-law phase indicates that CL-ZnMb/b5 has a variety of ZnMb−Cytb5 orientations for the facile ET in which the distance between the redox centers (D−A distance) is distributed over 13−20 Å. The single-exponential phase in the 3ZnMb* decay kinetics of CL-ZnMb/b5 is similar to the intrinsic decay of 3ZnMb* in its rate constant, 65 s-1. This implies that the ET is impeded in about 70% of the total ZnMb−Cytb5 orientations due to the D−A distance larger than 20 Å. Combined with the results of the Brownian dynamics simulations for the encounter complexes, the overall bimolecular ET rate, kapp, can be reproduced by the sum of the ET rates for the minor encounter complexes of which D−A distance is less than 20 Å. On the other hand, the encounter complexes with longer D−A distance, which are the majority of the encounter complexes between ZnMb and Cytb5, have little contribution to the overall bimolecular ET rate. These observations experimentally demonstrate that ZnMb forms a variety of encounter complexes with Cytb5, among which a minor set of the complexes with the shorter D−A distance (<∼20 Å) regulates the overall bimolecular ET between the proteins.

This publication has 30 references indexed in Scilit: