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

摘要

Site-directed mutagenesis has been used to study the effects of mutations resulting in surface charge changes near the Fe(Cys)(4) site of Clostridium pasteurianum rubredoxin (Rd). As predicted by simple electrostatics considerations, Rd variants with positively charged arginine residues in place of neutral surface residues ([V8R] and [L41R]) exhibit significant increases in the Fe(II/III) reduction potential. Contrary to electrostatics predictions, [V8D] and [V41D] Rd variants also exhibit significant increases in the Fe(II/III) reduction potential. These results indicate that protein electrostatic effects do not dominate as determinants of metal-site reduction potential in C. pasteurianum Rd. 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 experimental tests of this hypothesis are discussed. [References: 41]