PREPARATION, CHARACTERIZATION, AND INTRAMOLECULAR ELECTRON-TRANSFER IN PENTAAMMINERUTHENIUM HISTIDINE-26 CYTOCHROME-B5 DERIVATIVES - ROLE OF THE INTERVENING MEDIUM IN LONG-RANGE DONOR-ACCEPTOR ELECTRONIC COUPLING

Wild-type, mutant, and deuteroporphyrin-substituted bovine cytochromes b5 have been modified with pentaammineruthenium (a5Ru) for intramolecular electron-transfer (ET) studies. The reactivity of the three surface histidines of the wild-type trypsin-solubilized protein (Tb5) with [a5Ru(OH2)]2+ increases in the order His-15 < His-80 < His-26. Intramolecular ET rates from Fe(II) to Ru(III) have been measured by flash photolysis for a5Ru(His-26)-modified Tb5, Ru(H26)Tb5; mutant (Asn-57 to Asp, Gln-13 to Glu, Glu-11 to Gln, His-15 to Asn, His-80 to Asn) lipase-solubilized cyt b5, Ru(H26)LMb5; and deuteroporphyrin-substituted Tb5, Ru(H26)DPb5 (0.5-3-mu-M protein, mu = 0.5 M sodium phosphate, pH 7.0, 25-degrees-C). k(ET): 1.4 (1) s-1, Ru(H26)Tb5; 5.9 (5) s-1, Ru(H26)LMb5; 0.2 (1) s-1, Ru(H26)DPb5. The rates do not directly correspond to differences in driving force (-0.08, -0.10, -0.13 eV) or edge-to-edge donor-acceptor separation (12.1, 12, 12.9 angstrom). Evaluation of the donor-acceptor electronic coupling (H(AB)) in terms of specific through-bond and through-space interactions in the intervening medium for Ru(H26)Tb5 and Ru(H26)LMb5 revealed a probable ET pathway consisting of eight covalent bonds from C-gamma of His-26 to the end of the Leu-25 side chain and then a through-space jump (3.8, 3.7 angstrom) to the heme 2-vinyl. Ru(H26)DPb5 lacks the 2-vinyl, requiring a longer jump (4.5 angstrom) to the heme 3-methyl. Because H(AB) is predicted to decay rapidly with distance, the calculated rate constants (assuming-lambda = 1.2 eV) reflect the differences in the through-space jumps for the three derivatives: Ru(H26)Tb5, 1.9 s-1, Ru(H26)LMb5, 4.4 s-1; Ru(H26)DPb5, 0.16 s-1. The close agreement of calculated and observed rate constants indicates that the electronic coupling in this system is reasonably described by the His-26 --> Leu-25 --> through-space jump to heme pathway.