| 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) andcytochrome b5 (Cytb5) to reveal the mechanism of the inter-protein ET reactions under the condition ofmultiple encounter complexes. A variety of the ZnMb−Cytb5 orientations was suggested because of failureto identify the single and specific cross-linking site on Cytb5 by the peptide-mapping analysis using massspectrometry. 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 avariety of ZnMb−Cytb5 orientations for the facile ET in which the distance between the redox centers (D−Adistance) 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 isimpeded 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 overallbimolecular ET rate, kapp, can be reproduced by the sum of the ET rates for the minor encounter complexesof which D−A distance is less than 20 Å. On the other hand, the encounter complexes with longer D−Adistance, which are the majority of the encounter complexes between ZnMb and Cytb5, have little contributionto the overall bimolecular ET rate. These observations experimentally demonstrate that ZnMb forms avariety of encounter complexes with Cytb5, among which a minor set of the complexes with the shorterD−A distance (<∼20 Å) regulates the overall bimolecular ET between the proteins.
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