The loop region covering the iron-sulfur cluster in bovine adrenodoxin comprises a new interaction site for redox partners.

The amino acid in position 49 in bovine adrenodoxin is conserved among vertebrate [2Fe-2S] ferredoxins as hydroxyl function. A corresponding residue is missing in the cluster-coordinating loop of plant-type [2Fe-2S] ferredoxins. To probe the function of Thr-49 in a vertebrate ferredoxin, replacement mutants T49A, T49S, T49L, and T49Y, and a deletion mutant, T49Delta, were generated and expressed in Escherichia coli. CD spectra of purified proteins indicate changes of the [2Fe-2S] center geometry only for mutant T49Delta, whereas NMR studies reveal no transduction of structural changes to the interaction domain. The redox potential of T49Delta (-370 mV) is lowered by approximately 100 mV compared with wild type adrenodoxin and reaches the potential range of plant-type ferredoxins (-305 to -455 mV). Substitution mutants show moderate changes in the binding affinity to the redox partners. In contrast, the binding affinity of T49Delta to adrenodoxin reductase and cytochrome P-450 11A1 (CYP11A1) is dramatically reduced. These results led to the conclusion that Thr-49 modulates the redox potential in adrenodoxin and that the cluster-binding loop around Thr-49 represents a new interaction region with the redox partners adrenodoxin reductase and CYP11A1. In addition, variations of the apparent rate constants of all mutants for CYP11A1 reduction indicate the participation of residue 49 in the electron transfer pathway between adrenodoxin and CYP11A1.