Methods to fine-tune the rate of a fast conformational electron transfer (ET) gate involving a His-heme alkaline conformer of iso-1-cytochrome c (iso-1-Cytc) and to adjust the pH accessibility of a slow ET gate involving a Lys-heme alkaline conformer are described. Fine-tuning the fast ET gate employs a strategy of making surface mutations in a substructure unfolded in the alkaline conformer. To make the slow ET gate accessible at neutral pH, the strategy involves mutations at buried sequence positions which are expected to more strongly perturb the stability of native versus alkaline iso-1-Cytc. To fine-tune the rate of the fast His 73-heme ET gate, we mutate the surface-exposed Lys 79 to Ala (A79H73 variant). This mutation also simplifies ET gating by removing Lys 79, which can serve as a ligand in the alkaline conformer of iso-1-Cytc. To adjust the pH accessibility of the slow Lys 73-heme ET gate, we convert the buried side chain Asn 52 to Gly and also mutate Lys 79 to Ala to simplify ET gating (A79G52 variant). ET kinetics is studied as a function of pH using hexaammineruthenium(II) chloride (a(6)Ru(2+)) to reduce the variants. Both variants show fast direct ET reactions dependent on [a(6)Ru(2+)] and slower gated ET reactions that are independent of [a(6)Ru(2+)]. The observed gated ET rates correlate well with rates for the alkaline-to-native state conformational change measured independently. Together with the previously reported H73 variant (Baddam, S.; Bowler, B. E. J. Am. Chem. Soc. 2005, 127, 9702 - 9703), the A79H73 variant allows His 73-heme-mediated ET gating to be fine-tuned from 75 to 200 ms. The slower Lys 73 -heme (15-20 s time scale) ET gate for the A79G52 variant is now accessible over the pH range 6-8.