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

Abstract

A Pathways analysis of the methylamine dehydrogenase–amicyanin–cytochrome c-551i protein electron transfer (ET) complex predicts two sets of ET pathways 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 Cys 92 copper ligand, and in the other, it exits via the Met 98 copper ligand. 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 least 100-fold more strongly coupled than the pathways via any of the other copper ligands. All of the favored pathways via Cys 92 include a through-space 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 changed to other amino acid residues by site-directed mutagenesis. Some mutant proteins were very unstable suggesting a role for Tyr 30 in stabilizing the protein structure. Y30F and Y30I mutant amicyanins could be isolated and analyzed. For the Y30I mutant, the modified Pathways analysis which favors 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 amicyanin. Likely explanations for these observations are discussed as are their implications for predicting pathways for ET reactions of metalloproteins.