Tyr(30) of amicyanin is not critical for electron transfer to cytochrome c-551i: implications for predicting electron transfer pathways

A Pathways analysis of the methylamine dehydrogenase-amicyanin-cytochrome c -551i protein electron transfer (ET) complex predicts two sets of ET pathwa ys of comparable efficiency from the type I copper of amicyanin to the heme of cytochrome c-551i. In one pathway, the electron exits copper via the Cy s(92) copper ligand, and in the other, it exits via the Met(98) copper liga nd. If the Pathways algorithm is modified to include contributions from the anisotropy of metal-ligand coupling, independent of differences in copper- ligand bond length, then the pathways via Cys(92) are predicted to be at le ast 100-fold more strongly coupled than the pathways via any of the other c opper ligands. All of the favored pathways via Cys(92) include a through-sp ace jump from Cys(92) to the side chain of Tyr(30). To determine whether or not the pathways via Cys(92) are preferentially used for ET, Tyr(30) was c hanged to other amino acid residues by site-directed mutagenesis, Some muta nt proteins were very unstable suggesting a role for Tyr(30) in stabilizing the protein structure. Y30F and Y30I mutant amicyanins could be isolated a nd analyzed. For the Y30I mutant, the modified Pathways analysis which favo rs ET via Cys(92) predicts a decrease in ET rate of at least two orders of magnitude, whereas the standard Pathways analysis predicts no change in ET rate since ET via Met(98) is not affected. Experimentally, the ET rates of the Y30I and Y30F mutants were indistinguishable from that of wild-type ami cyanin, Likely explanations for these observations are discussed as are the ir implications for predicting pathways for ET reactions of metalloproteins , (C) 2000 Elsevier Science B.V, All rights reserved.