We have investigated the photoinduced electron transfer (ET) in the 1: 1 cross-linked complex (CL-ZnMb/b(5)) formed by a cross-linking reagent, EDC, between Zn-substituted myoglobin (ZnMb) and cytochrome b(5) (Cytb(5)) to reveal the mechanism of the inter-protein ET reactions under the condition of multiple encounter complexes. A variety of the ZnMb-Cytb(5) orientations was suggested because of failure to identify the single and specific cross-linking site on Cytb(5) by the peptide-mapping analysis using mass spectrometry. In CL-ZnMb/b(5), a laser pulse generates the triplet excited state of the ZnMb domain ((3)ZnMb(*)), which can transfer one electron to the Cytb(5) domain. The decay kinetics of (3)ZnMb(*) in CL-ZnMb/b(5) consists of a facile power-law ET phase to Cytb(5) domain (similar to30%) and a slower single-exponential phase (similar to70%). The application of the Marcus equation to this power-law phase indicates that CL-ZnMb/b(5) has a variety of ZnMb-Cytb(5) orientations for the facile ET in which the distance between the redox centers (D-A distance) is distributed over 13-20 Angstrom. The single-exponential phase in the (3)ZnMb(*) decay kinetics of CL-ZnMb/b(5) is similar to the intrinsic decay of (3)ZnMb(*) in its rate constant, 65 s(-1), This implies that the ET is impeded in about 70% of the total ZnMb-Cytb(5) orientations due to the D-A distance larger than 20 Angstrom. Combined with the results of the Brownian dynamics simulations for the encounter complexes, the overall bimolecular ET rate, k(app), can be reproduced by the sum of the ET rates for the minor encounter complexes of which D-A distance is less than 20 Angstrom. On the other hand, the encounter complexes with longer D-A distance, which are the majority of the encounter complexes between ZnMb and Cytb(5), have little contribution to the overall bimolecular ET rate. These observations experimentally demonstrate that ZnMb forms a variety of encounter complexes with Cytb(5), among which a minor set of the complexes with the shorter D-A distance < &SIM;20 &ANGS;) regulates the overall bimolecular ET between the proteins.