Synergistic Effects of Copper Sites on Apparent Stability of Multicopper Oxidase, Fet3p

Int J Mol Sci. 2018 Jan 16;19(1):269. doi: 10.3390/ijms19010269.

Abstract

Saccharomyces cerevisiae Fet3p is a multicopper oxidase that contains three cupredoxin-like domains and four copper ions located in three distinct metal sites (T1 in domain 3; T2 and the binuclear T3 at the interface between domains 1 and 3). To probe the role of the copper sites in Fet3p thermodynamic stability, we performed urea-induced unfolding experiments with holo-, apo- and three partially-metallated (T1, T2 and T1/T2 sites depleted of copper) forms of Fet3p. Using a combination of spectroscopic probes (circular dichroism, fluorescence intensity and maximum, 8-anilinonaphthalene-1-sulfonic acid (ANS) emission, oxidase activity and blue color), we reveal that all forms of Fet3p unfold in a four-state reaction with two partially-folded intermediates. Using phase diagrams, it emerged that Fet3p with all copper sites filled had a significantly higher stability as compared to the combined contributions of the individual copper sites. Hence, there is long-range inter-domain communication between distal copper sites that contribute to overall Fet3p stability.

Keywords: Fet3p; cupredoxin-like domain; multicopper oxidases MCO; multidomain protein stability; phase diagram method.

MeSH terms

  • Apoproteins / metabolism
  • Ceruloplasmin / metabolism*
  • Copper / metabolism*
  • Enzyme Stability
  • Mutant Proteins / metabolism
  • Protein Denaturation / drug effects
  • Protein Folding / drug effects
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Spectrometry, Fluorescence
  • Urea / pharmacology

Substances

  • Apoproteins
  • Mutant Proteins
  • Saccharomyces cerevisiae Proteins
  • Copper
  • Urea
  • Ceruloplasmin
  • FET3 protein, S cerevisiae