The essential role of Glu-185 and Tyr-354 residues in the ferroxidase activity of Saccharomyces cerevisiae Fet3

The structural determinants required for ferroxidase activity by the yeast multicopper oxidase Fet3 have been partially clarified by site-directed mut agenesis based on homology modeling. Glu-185 and Tyr-354 mere substituted w ith Ala and Phe, respectively, Fet3 E185A retained ca, 5% residual ferroxid ase catalytic efficiency, and almost 40% oxidase efficiency. On the other h and, Fet3 Y354F exhibited 50% residual efficiency as a ferroxidase and more than 70%, as an oxidase, These results provide new insights in the mechani sm of iron binding and oxidation by Fet3, establishing the essential role o f Glu-185 and Tyr-354, and allowing to dissect ferroxidase from non-iron ox idase activity. (C) 2000 Federation of European Biochemical Societies.