The vertebrate ferredoxins are 2Fe-2S proteins found in the matrix of mitochondria of steroid metabolizing tissues. They function in electron transfer from NADPH-oxidoreductases to cytochromes P450 involved in steroid hormone, vitamin D, and bile acid metabolism. We propose to carry out functional and structural studies on recombinant human ferredoxin produced in E. coli. Site-directed mutagenesis methods of protein engineering will be used to 1) change amino acids involved in metal coordination and create new iron- sulfur clusters having different ligands, 2) investigate the pH dependence of the oxidation-reduction potential by altering charged amino acids near the 2Fe-2S center, and 3) identify residues involved in binding and electron transfer with NADPH- oxidoreductase and cytochromes P450scc and P450-11Beta. In addition, attempts will be made to obtain crystals of both wild type and mutant forms of recombinant ferredoxin for X-ray diffraction analysis.
