In order to understand the roles of protein matrix in electron transfer processes (ET) within biological systems, a heme-based donor (Zn-heme: ZnPP)-sensitizer (Ru2+(bpy)(3))-acceptor (cyclic viologen: BXV4+) triad 1 was used as a probe molecule. Two semisynthetic systems, Cyt-b(562)(1) and Mb(1), in which the triad is incorporated into cytochrome b(562) (Cyt-b(562)) or into myoglobin (Mb), were constructed by cofactor reconstitution. These two semisynthetic proteins were compared with the triad itself (i.e., without the protein matrix) using absorption spectroscopy, steady state emission and excitation studies, laser flash photolysis experiments, and molecular modeling. Photoexcitation of the ZnPP moiety of Cyt-b(562)(1) or Mb(1) leads to a direct ET from the triplet state of ZnPP state ((ZnPP)-Zn-3) to BXV4+ to generate a final charge-separated (CS) state, Cyt-b(562)(Zn+)-Ru2+-BXV3+. or Mb(Zn+)-Ru2+-BXV3+.. On the other hand, direct ET from the excited ZnPP moiety to the BXV4+ moiety is also involved in 1 in the absence of the protein matrix, but the excited state of ZnPP involved is not (ZnPP)-Zn-3, but the singlet excited state ((ZnPP)-Zn-1) in this pathway. When the Ru2+(bpy)(3) moiety of Cyt-b(562)(1) or Mb(1) is excited, a stepwise ET relay occurs with the ion-pair, Cyt-b(562)(Zn)Ru3+-BXV3+. or Mb(Zn)-Ru3+-BXV3+. as an intermediate, leading to the same final CS state as that generated in the direct ET pathway. The lifetimes of the corresponding final CS states were determined to be 300 ns for 1 in the absence of the protein matrix, 600-900 ns for Cyt-b(562)(1) and 1.1-18 mu s for Mb(1), the values of which are greatly affected by the protein matrix. Molecular modeling study of the three systems consistently explained the differences of their photophysical behavior.