W0079

Structural Studies of Amicyanin Mutants That Stabilize the Reduced State of Copper. Christopher Carrell, Scott Mathews, Victor Davidson, Dapeng Sun, Dept. of Biochemistry and Molecular Biophysics, Washington Univ. School of Medicine, 660 S. Euclid St. Box 8231, St. Louis, MO 63110 USA.

High resolution structures (dmin < 1.1 Å) of oxidized and reduced amicyanin mutants P94F and P94A have been obtained. The P94F mutant crystallizes in the orthorhombic space group P212121, a=51.4 Å, b=59.7 Å, c=74.8 Å, _=_=_=90_, with two molecules in the asymmetric unit. The P94A mutant is isomorphous with wild-type amicyanin in space group P21, a=28.4 Å, b=55.4 Å, c=27.1 Å, _=95.2_, _=_=90_. All data were collected at Beamline 14-BM-C of BIOCARS at the Advanced Photon Source, Argonne, IL.

Amicyanin is an obligatory intermediate in electron transfer between methylamine dehydrogenase (MADH) and cytochrome c551i in Paracoccus denitrificans with a midpoint potential of +300 mV at pH 7. The P94F and P94A mutants of amicyanin have midpoint potentials of +400 mV and +380 mV at pH 7, respectively (Machczynski et al. J. Inorg. Biochem. 2000, p. 375-380). The increase in midpoint potential as a result of these mutations at Pro94 arises from a stabilization of the reduced state of copper through the addition of a hydrogen bond from the backbone at residue 94 to the copper-coordinating sulfur atom of Cys92.

The resulting structures of amicyanin with site-directed mutations at Pro94 lead to two distinct observations. Oxidized and reduced P94F amicyanin at pH 5.5 appear to be isostructural to each other. In reduced P94A amicyanin at pH 5.5, on the other hand, there appear to be two partially occupied copper sites separated by 1.4 Å, a result that is not observed in wild-type amicyanin.