TUNING THE REDOX POTENTIAL OF AN ELECTRON TRANSFER PROTEIN: ANALYSIS OF THE HIGH POTENTIAL P80I PSEUDOAZURIN C.A. Peters-Libeu¹, E.T. Adman¹, S. Turley¹, T. Beppu ² and M. Nishiyama². ¹Department of Biological Structure, University of Washington, Box 35742, Seattle, WA. , USA 98195. ²Department of Biotechnology and Biotechnology Research Center, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113. Japan

The P80I mutant of Alcaligenes faecalis pseudoazurin has the largest redox potential change (+150mV) observed for a non-ligand single site cupredoxin mutant. In pseudoazurin, the copper is in a distorted tetrahedral coordination with two histidines, a cysteine and a methionine. Reduction of native and P80A pseudoazurin at physiological pH induces a lengthening of the methionine copper bond which results in a more trigonal coordination of the copper. Structural analysis of the oxidized and reduced forms of the P80I mutant has revealed that in both oxidized and reduced P80I pseudoazurin the copper methionine bond length is equivalent to the copper methionine bond lengths observed for reduced native and P80A pseudoazurin The flattened copper center in oxidized P80I pseudoazurin is very similar to the copper center found in the oxidized form of the very high potential cupredoxin Rusticyanin. The large increase in the redox potential observed for the P80I mutant may be due to a decrease in the reorganization energy of the copper center required for reduction. The ability of the copper center in P80I pseudoazurin to adopt this stable flattened coordination appears to result from an alteration of the hydrogen bond network surrounding the copper site. In cupredoxins, this hydrogen bond network has been suggested to stabilize the copper center in its characteristic geometry.