Artificial photosynthetic reaction centers have been constructed on a protein surface by cofactor reconstitution, which mimic the function of photosynthetic organisms to convert light energy to chemical potential in the form of long-lived charge-separated stairs. They feature a ruthenium tris(2,2'-bipyridine) moiety as the sensitizer, which is mechanically linked (i.e., in catenane-type) with a cyclobis(paraquat-p-phenylene) unit (BXV4+, acceptor) and covalently linked with a protoheme or Zn-protoporphyrin (donor) located in the myoglobin pocket. Their cofactors 1 and 2, which are tris(heteroleptic) Ru-bipyridine complexes, were synthesized by sequential coordination of the two different functionalized bipyridine ligands with a readily obtainable precursor [Ru(4,4'-dimethyl-2,2'-bipyridine)Cl-3](n) followed by metal insertion; this represents a new efficient synthetic method for tris(heteroleptic) Ru(TI) complexes of bidentate polypyridine ligands. Reconstitution of apo-myoglobin (Mb) with 1 and 2 affords the two Mb-based artificial triads, Mb((FeOH2)-O-III)-Ru2+-BXV4+ and Mb(Zn)-Ru2+-BXV4+. Laser flash photolysis of the Ru(bpy)(3) moiety of Mb((FeOH2)-O-III)-Ru2+-BXV4+ in an aqueous solution yields an initial charge-separated state, Mb((FeOH2)-O-III .)-Ru2+-BXV3+., via noncovalent electron transfer, followed by dark electron transfer to generate an intermediate consisting of porphyrin cation radical, Mb((FeOH2)-O-III .)-Ru2+-BXV3+.. Mb((FeOH2)-O-III .)-Ru2+-BXV3+. thus generated is subsequently converted, via a proton-coupled process and with a quantum yield of 0.005, into the final charge-separated state, Mb(Fe-IV=O)-Ru2+-BXV3+., which bears an energy more than 1 eV above the ground state and a lifetime (tau > 2 ms) comparable to that of natural photosynthetic reaction center. Photoexcitation of Mb(Zn)-Ru2+-BXV4+ also gives rise to a vectorial two-step electron-transfer relay with the intermediate CS state, Mb(Zn)-Ru3+-BXV3+., for the main pathway leading to the final CS state, Mb(Zn+)-Ru2+-BXV3+., in a yield of 0.08. Although the driving forces for the recombination of Mb(Fe-IV=O)-Ru2+-BXV3+. and Mb(Zn+)-Ru2+-BXV3+. are similar (Delta G approximate to 1.30 eV), the recombination rate of the former is at least 10(2)-10(3)-fold slower than that of the latter. By analogy with a related system reported previously, it was considered that back ET from BXV3+. to Mb(Fe-IV=O) might be coupled to the protonation of Mb(Fe-IV=O) and governed by the slow interconversion between the metal-oxo form and the proton-activated species, rendering the CS state Mb(Fe-IV=O)-Ru2+-BXV3+. specially long-lived. Control experiments clearly demonstrated that partial incorporation of the triads into the protein matrix plays a crucial role in regulating the electron-transfer pathway and stabilizing the charge separation state.