Toluene 4-monooxygenase (T4MO) catalyzes the NADH- and O₂-dependent hydroxylation of toluene to form p-cresol. In this four-protein complex, T4moF transfers electrons to T4moC, a Rieske-type ferredoxin and T4moC transfers electrons to T4moH, a diiron hydroxylase. T4moD is a catalytic effector that increases the rate and efficiency of reaction. Both T4moC and T4moD form protein-protein complexes with T4moH. These can only be substituted by proteins from within the same evolutionary clades, suggesting unique specificities of the interactions required for catalysis. The 1.48 Å X-ray structure of T4moC revealed a prominent negatively charged surface near to the [2Fe-2S] cluster. In addition, five hydrogen bonds to the Cys-SG and bridging sulfur atoms of the [2Fe-2S] cluster were present, suggesting a positive redox potential (0 mV or greater versus NHE). The closest structural homolog to T4moC, the BphF Rieske ferredoxin from biphenyl dioxygenase, has only three hydrogen bonds and E˚' ≈ -150 mV. This combination of properties warranted a more detailed examination of the role of active site residues and redox potentials. In T4moC, Trp69 is adjacent to the [2Fe-2S] center in a comparable position to a serine that contributes an additional hydrogen bond in the respiratory complex Rieske ferredoxins (E˚' ≈ +150 to +350 mV). Saturation mutagenesis of W69 revealed that only W69F and W69Y retained significant catalytic activity, suggesting the importance of an aromatic residue in this position. The E˚' of T4moC was -173mV, while W69F T4moC had E˚' of -137mV. TbuB, a closely related Rieske ferredoxin, has the comparable Trp residue and E˚' of -150mV. These E˚' values are significantly lower than expected from the number of hydrogen bonds to the [2Fe-2S] cluster and suggest that total surface charge and electron density adjacent to a cluster sulfide from Trp69 contribute to the lowered redox potential. Funded by NSF MCB-0316232 to B.G.F.