The amino acid in position 54 of adrenodoxin is strongly conserved among ferredoxins, consisting of a threonine or serine. Its role was studied by analyzing mutants T54S and T54A of bovine adrenodoxin. Absorption, circular dichroism, fluorescence, and electron paramagnetic resonance spectra of mutant T54S show that this substitution has no influence on the formation and stability of the ferredoxin. The redox potential of this mutant, however, was lowered by 55 mV as compared with native adrenodoxin, indicating a role for this residue in redox potential modulation. Incorporation of the iron-sulfur cluster was not impaired in the T54A mutant, although structural features of the oxidized protein were considerably changed. The decreased stability of the T54A mutant as compared with the wild type and mutant T54S indicates that a hydrogen bond donor at this position stabilizes the protein. Both mutants have been shown to be functionally active. Replacement of threonine 54 by serine or alanine, however, leads to rearrangements at the recognition sites for its redox partners. This is reflected by decreased Km and Kd values of both mutants for the cytochromes P450, whereas only T54A displayed a decreased Km value in cytochrome c reduction. Substrate conversion was accelerated (2.2- and 2.4-fold for mutants T54A and T54S, respectively) in the CYP11B1-, but not in the CYP11A1-dependent reaction.