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

Abstract

The structural determinants required for ferroxidase activity by the yeast multicopper oxidase Fet3 have been partially clarified by site-directed mutagenesis based on homology modeling. Glu-185 and Tyr-354 were substituted with Ala and Phe, respectively. Fet3 E185A retained ca. 5% residual ferroxidase catalytic efficiency, and almost 40% oxidase efficiency. On the other hand, Fet3 Y354F exhibited 50% residual efficiency as a ferroxidase and more than 70% as an oxidase. These results provide new insights in the mechanism of iron binding and oxidation by Fet3, establishing the essential role of Glu-185 and Tyr-354, and allowing to dissect ferroxidase from non-iron oxidase activity.

A Novel Extracellular Multicopper Oxidase from Phanerochaete chrysosporium with Ferroxidase Activity

“…In contrast, rMCO1 had a high level of ferroxidase activity, with a K m on the same order of magnitude as that described for Fet3 (11). In addition, considerable oxidase activity with aromatic amines was observed, a property common among Fet3 proteins (7,11). On the other hand, the optimum pH of rMCO1 (pH 3.4) is lower than the optimum pHs of Fet3 family members, which are near pH 5.0 (11).…”

“…Several studies have focused on identification of the structural determinants that confer ferroxidase activity on MCOs (2,7,8). Glu-185 and Tyr-354 are essential for the oxidation of Fe 2ϩ by Fet3 from S. cerevisiae.…”

The copper-iron connection in biology: Structure of the metallo-oxidase Fet3p

“…The Fet3p structure places these residues on the protein surface, distant from both the T1 and trinuclear sites. Two groups have investigated the activity of mutants constructed at E185 and D278 (and Y354) (23,61) and to these we add D283 and D409. All of these were placed at or near the T1 Cu by homology modeling, and the structure reveals that the side chains of E185, D283, and D409 are oriented toward the T1 Cu, whereas the side chains of D278 and Y354 are not.…”

Characterization of PIR1, a GATA family transcription factor involved in iron responses in the white-rot fungus Phanerochaete chrysosporium

“…The same observation is valid for the genes encoding for MCO2 and MCO4. Particularly intriguing in the case of the latter enzyme, multiple alignments of all MCOs have shown the presence of a Glu residue equivalent to that of the Fet3 from Saccharomyces cerevisiae involved in iron oxidation (Bonaccorsi di Patti et al, 2000), with the sole exception of MCO4 (Larrondo et al, 2003(Larrondo et al, , 2007a.…”