Mutational analysis of the iron binding site of Saccharomyces cerevisiae ferroxidase Fet3. An in vivo study.
The role of residues predicted to be involved in the binding of iron by the yeast ferroxidase Fet3 has been studied by site-directed mutagenesis. The effect of Fet3 mutations E185A, E185Q, Y354F, D409V and H489D has been investigated in vivo by kinetic analyses of high affinity iron uptake. Our results indicate that Glu-185 is critical for the binding of iron, since substitution of this residue with Ala or Gln strongly affects both growth and the kinetic parameters of high affinity iron uptake, greatly increasing K(m). Mutations Y354F and D409V result in less severe alteration of high affinity iron uptake, while mutant H489D is unable to grow under conditions of iron limitation.
Bonaccorsi di Patti
M C
MC
Department of Biochemical Sciences, University of Rome La Sapienza, Italy.
Paronetto
M P
MP
Dolci
V
V
Felice
M R
MR
Lania
A
A
Musci
G
G
D019943:Amino Acid Substitution; D001665:Binding Sites; D002570:Ceruloplasmin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002712:Chlorides; D004252:DNA Mutational Analysis; D005290:Ferric Compounds / Q000378:metabolism*; D005816:Genetic Complementation Test; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic
D002712:Chlorides; D005290:Ferric Compounds; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D002570:Ceruloplasmin; C085513:FET3 protein, S cerevisiae; C024555:ferric chloride