The iron in the heme group of horse-heart cytochrome c was replaced by cobalt according to established methods. The resulting cobalticytochrome c was subsequently modified at histidine-33 with a pentaammineruthenium group. Proof of correct derivatization was obtained by atomic absorption analysis of cobalt and ruthenium, differential pulse voltammetry, and enzymatic proteolysis analyzed by diode-array HPLC. Cobalt(II)-to-ruthenium(III) intramolecular electron transfer rates were measured as a function of temperature by electron pulse radiolysis. The azide radical (N-3(.)) was used to oxidize the fully reduced form in order to generate the desired electron transfer precursor. The intramolecular electron transfer rate is 1.28 +/- 0.04 s(-1) at 25 degrees C (Delta H double dagger = 5.7 +/- 0.2 kcal/mol, Delta S double dagger = -38.7 +/- 0.5 cal/(deg mel)) for a driving force of 0.28 +/- 0.02 eV. The results are compared with those for analogous pentaammineruthenium-modified, native iron, and zinc-substituted cytochromes c. The 0.4 eV increase in driving force for intramolecular electron transfer when iron is replaced by cobalt is largely compensated by an increase in reorganization energy.