Structure-function analysis of the cuprous oxidase activity in Fet3p from Saccharomyces cerevisiae.
The Fet3 protein from Saccharomyces cerevisiae is a multicopper oxidase with specificity toward Fe(II) and Cu(I). Fet3p turnover of Fe(II) supports high affinity iron uptake across the yeast plasma membrane, whereas its turnover of Cu(I) contributes to copper resistance in yeast. The structure of Fet3p has been used to identify possible amino acid residues responsible for this protein's reactivity with Cu(I), and structure-function analyses have confirmed this assignment. Fet3p Met(345) is required for the enzyme's reactivity toward Cu(I). Although the Fet3pM345A mutant exhibits wild type spectral and electrochemical behavior, the kinetic constants for Cu(I) turnover and for single-turnover electron transfer from Cu(I) to the enzyme are significantly reduced. The specificity constant with Cu(I) as substrate is reduced by one-fifth, whereas the electron transfer rate from Cu(I) is reduced 50-fold. This mutation has little effect on the reactivity toward Fe(II), indicating that Met(345) contributes specifically to Fet3p reactivity with the cuprous ion. These kinetic defects render the Fet3pM345A unable to support wild type cellular copper resistance, suggesting that there is a finely tuned copper redox balance at the yeast plasma membrane.
Stoj
Christopher S
CS
Department of Biochemistry, School of Medicine and Biomedical Sciences, The University at Buffalo, Buffalo, New York 14214, USA.
Augustine
Anthony J
AJ
Solomon
Edward I
EI
Kosman
Daniel J
DJ
D002462:Cell Membrane / Q000378:metabolism; D002570:Ceruloplasmin / Q000737:chemistry* / Q000502:physiology*; D003300:Copper / Q000737:chemistry; D018360:Crystallography, X-Ray; D004563:Electrochemistry; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D008715:Methionine / Q000737:chemistry; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D010088:Oxidoreductases / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000502:physiology*; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays
D029701:Saccharomyces cerevisiae Proteins; D003300:Copper; D008715:Methionine; D007501:Iron; D010088:Oxidoreductases; D002570:Ceruloplasmin; C085513:FET3 protein, S cerevisiae