SITE-DIRECTED MUTAGENESIS OF RUBREDOXIN REVEALS THE MOLECULAR-BASIS OF ITS ELECTRON-TRANSFER PROPERTIES

Rubredoxins contain a single non-heme iron atom coordinated by four cy steines. This iron is redox active and confers a role to these protein s in electron transfer chains. The structural features responsible for setting the values of the reduction potential and of the electron sel f-exchange rate constant have been probed by site-directed mutagenesis . Replacements of the highly conserved residues in positions 8, 10, an d 11 (valine, glycine, and tyrosine, respectively) all lead to shifts of the reduction potential, up to 75 mV. These cannot be explained by simple considerations about the physicochemical properties of the subs tituting side chains but rather indicate that the value of the reducti on potential is finely tuned by a variety of interactions. In contrast , the electron self-exchange rate constant measured by nuclear magneti c resonance does not vary much, except when a charged residue is inclu ded in position 8 or 10, at the surface of the protein closest to the iron atom. Analysis of the data with a model for electrostatic interac tions, including both monopolar and dipolar terms, indicates that the presence of a charge in this region not only increases the repulsion b etween molecules but also affects the electron transfer efficiency of the bimolecular complexes formed. The studies presented constitute a f irst step toward probing the structural elements modulating the reacti vity of the FeS4 unit in a protein and defining the electron transfer active site(s) of rubredoxin.