Ruthenium bisbipyridine complexes of horse heart cytochrome c: Characterization and comparative intramolecular electron-transfer rates determined by pulse radiolysis and flash photolysis

The reaction of [Ru(bpy)(2)L(H2O)](2+) (bpy = 2,2'-bipyridine, L = imidazol e, water) with reduced horse heart cytochrome c results in coordination of [Ru-II(bpy)(2)L] at the His 33 and His 26 sites. Coordination at the His 33 site gave a diastereomeric [Ru-II(bpy)(2)L]-His-cyt c(II) mixture favoring the Il-Ru form regardless of the substituent on the bipyridine ligands, wh ile substitution at the more buried His 26 site gave an isomeric distributi on that varies according to the substituent on the bipyridine ligands. The diastereomeric aquoproteins (L = H2O) are distinguished by their redox pote ntials and their conversion to the corresponding fluorescent imidazole prot eins. Intramolecular electron transfer between the reduced ruthenium bipyri dine and cyt c(III) in [Ru-II(bpy(.))(bpy)L]-His33-cyt c(II) was determined by reductive pulse radiolysis using the aqueous electron as a reducing age nt, k(ret) = (2.0 +/- 0.3) x 10(5) s(-1), and k(ret) is independent of the sixth ligand L = H2O imidazole. In addition, the rate constant for intramol ecular electron transfer from cyt c(II) to the ruthenium(III) center in [Ru -III(bpy)(2)L]-His33-cyt c(II)) was determined by oxidative pulse radiolysi s using azide and carbonate radicals. This rate is very sensitive to the na ture of the sixth ligand. When L = H2O, the intramolecular electron-transfe r rate for the major diastereomer Lambda-cis-[Ru-III (bpy)(2)(H2O)]-His33-c yt c(II) is k = 1.1 x 10(4) s(-1) and is independent of pH between 5.6 and 8.3. The minor Delta-cis-[Ru-III(bpy)(2)(H2O)]-His33-cyt c(II) isomer has p H-dependent electrochemistry and a lower rate of intramolecular electron tr ansfer. Complete conversion from L = H2O to L = imidazole is slow, requirin g more than 7 days in 1 M imidazole. A lower limit (k > 2 x 10(6) s(-1)) fo r the intramolecular electron-transfer rate constant in [Ru-III(bpy)(2)(L)] -His33-cyt c(II), L = imidazole, could be obtained by pulse radiolysis in t he absence of the slower reacting aquo species. This observation is in agre ement with the value of 3 x 10(6) s(-1) measured by flash photolysis. Earli er pulse radiolysis experiments primarily measured the aquoligated rutheniu m protein, while the flash photolysis experiments measured the imidazole-li gated fraction because it is the only species oxidatively quenched in the p hotoinduced reactions. Intramolecular electron-transfer reactions for a new series of ruthenium bipyridine complexes, [Ru(dabpy)(2)L]-His33-cyt c prot eins (dabpy = 4,4'-diamino-2,2'-bipyridine ) (L imidazole, pyridine, isonic otinamide and pyrazine), proceed with lower driving force, resulting in slo wer rate constants amenable to measurement by oxidative pulse radiolysis. T he electron-transfer rate constants for this series spanned a wide range of the Marcus log k vs Delta G plot.