THE ROLE OF THREONINE-54 IN ADRENODOXIN FOR THE PROPERTIES OF ITS IRON-SULFUR CLUSTER AND ITS ELECTRON-TRANSFER FUNCTION

The amino acid in position 54 of adrenodoxin is strongly conserved amo ng ferredoxins, consisting of a threonine or serine. Its role was stud ied by analyzing mutants T54S and T54A of bovine adrenodoxin, Absorpti on, circular dichroism, fluorescence, and electron paramagnetic resona nce spectra of mutant T54S show that this substitution has no influenc e on the formation and stability of the ferredoxin. The redox potentia l of this mutant, however, was lowered by 55 mV as compared with nativ e adrenodoxin, indicating a role for this residue in redox potential m odulation, Incorporation of the iron-sulfur cluster was not impaired i n the T54A mutant, although structural features of the oxidized protei n were considerably changed. The decreased stability of the T54A mutan t as compared with the wild type and mutant T54S indicates that a hydr ogen 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 recogni tion sites for its redox partners. This is reflected by decreased K-m and K-d values of both mutants for the cytochromes P450, whereas only T54A displayed a decreased K-m value in cytochrome c reduction, Substr ate 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.