CRYSTAL STRUCTURES OF CLOSTRIDIUM PASTEURIANUM RUBREDOXIN MUTANTS V8A AND V8N. Tom J. Brett*, John J. Stezowski*, Kathy M. Selbo*, Charles R. Ross II*, Qiandong Zeng⁺, Donald M. Kurtz⁺, and Robert A. Scott⁺, *Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304 ⁺Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602-2556

Crystal structures of V8A and V8N mutants of Clostridium pasteurianum rubredoxin (Rd) have been determined at 1.68 and 1.82 resolution data giving final R-values of 14.10% and 14.40%, respectively. The structure determinations were pursued to enhance observations from recent studies in which redox potentials of several Rd mutants were determined¹. In these studies, site-directed mutagenesis was used to study the effects of mutations resulting in surface charge changes near the Fe(Cys)₄ site of Rd. As predicted by simple electrostatic considerations, Rd mutants with positively charged arginine residues in place of neutral surface residues (V8R and L41R) exhibit significant increases in the Fe(II/III) reduction potential. However, significant increases in reduction potential were also seen in mutants where the local negative charge was increased (V8D and L41D), and also when neutral residues were replaced by other neutral residues (V8A and V8N). These findings suggest that reduction potential changes are not dominated by electrostatic effects. Rather, it has been proposed that these reduction potential changes are due to increases in the local dielectric at the Fe(Cys)₄ center. Under this hypothesis, increasing the local dielectric, e.g., by increasing the solvent accessibility of the Fe(Cys)₄ site, would increase the reduction potential. In comparison with the Rd wild-type structure, the V8A and V8N mutants show increased solvent accessibility at one of the cysteine sulfurs (S-Cys42).

1 Q. Zeng, E.T. Smith, D.M. Kurtz, Jr., R.A. Scott. Inorg. Chim. Acta, in press.