PROTEIN DETERMINANTS OF METAL SITE REDUCTION POTENTIALS - SITE-DIRECTED MUTAGENESIS STUDIES OF CLOSTRIDIUM-PASTEURIANUM RUBREDOXIN

Site-directed mutagenesis has been used to study the effects of mutati ons resulting in surface charge changes near the Fe(Cys)(4) site of Cl ostridium pasteurianum rubredoxin (Rd). As predicted by simple electro statics considerations, Rd variants with positively charged arginine r esidues in place of neutral surface residues ([V8R] and [L41R]) exhibi t significant increases in the Fe(II/III) reduction potential. Contrar y to electrostatics predictions, [V8D] and [V41D] Rd variants also exh ibit significant increases in the Fe(II/III) reduction potential. Thes e results indicate that protein electrostatic effects do not dominate as determinants of metal-site reduction potential in C. pasteurianum R d. A hypothesis is developed that increased solvent accessibility and the resultant increase in polarity of the Fe(Cys)(4) site dominate as determinants of the reduction potential in this protein. Possible expe rimental tests of this hypothesis are discussed.