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 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.
Bonaccorsi di Patti
M C
MC
Department of Biochemical Sciences, University of Rome La Sapienza, Rome, Italy.
Felice
M R
MR
Camuti
A P
AP
Lania
A
A
Musci
G
G
D002384:Catalysis; D002570:Ceruloplasmin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D018698:Glutamic Acid; D014443:Tyrosine; D002570:Ceruloplasmin; C085513:FET3 protein, S cerevisiae