The rates of intramolecular electron-transfer (ET) reactions from the ferroheme to bis(2,2-bipyridine)(imidazole)-ruthenium(III) complexes bound to genetically engineered histidines (58 and 66) on the surface of yeast iso-1-cytochrome c (cyt c) have been measured by using a laser flash-quench technique. The crystal structure of the wild-type protein indicates that the ET pathways involve aromatic side chains : Ru(His58)cyt c includes a bridging tryptophan at position 59, and Ru(His66)cyt c has a tyrosine at 67. A variant in which the bridging Tyr67 in the His66 mutant had been replaced with a phenylalanine also was examined. The Fe2+ --> Ru3+ET rate constants (25-degrees-C, pH 7.0) are as follows : 5.2(5) X 10(4) (DELTAE-degrees = 0.69(5)), Ru(His58)cyt c; 1.0(1) X 10(6) (DELTAE-degrees = 0.72(5)), Ru(His66)cyt c; and 3.1(3) X 10(6) s-1 (DELTAE-degrees = 0.77(5) eV), Ru(His66Phe67)cyt c. The experimentally derived electronic coupling constants [H(AB)(His58) = 0.014; H(AB)(His66) = 0.060 cm-1] are in closer agreement with the lengths of sigma-tunneling pathways than with the direct donor-acceptor distances, and there is no indication that the pi orbitals of intervening groups enhance any of these couplings. Maximum ET rates in the modified cytochromes drop by 2 orders of magnitude for every 6.3-angstrom increase in the sigma-tunneling length. Analysis of the results also suggests that an internal water molecule in Ru(His66Phe67)cyt c plays a role in linking the Ru(His66) group to the heme.