Mutational analysis of substrate interactions and oxygen reduction of the multicopper oxidase Fet3p from Saccharomyces cerevisiae
Stoj, Christopher Stephen
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Metal ions such as iron, copper, and zinc are essential cofactors in many biochemical processes throughout nature. The critical requirement for metals in biology have been know for many years and is typified by the red pigment of human blood as a result of oxygen binding to the iron containing protein hemoglobin. Currently, altered metal ion regulation is considered a causative agent of many disease states, including those of neurodegenerative diseases such as Alzheimer's and Amyotrophic Lateral Sclerosis, also known as Lou Gehrig's disease. Investigations of cellular metal ion regulation are central to developing a significant understanding of roles metals play in various pathologies. The baker's yeast Saccharomyces cerevisiae serves as the model system to study eukaryotic iron and copper metallobiochemistry. In general, yeast protein components for the uptake and storage of iron and copper are quite similar to those found in various higher eukaryotes including human cells. One example of this homology is the yeast high-affinity iron uptake system composed of the multicopper ferroxidase Fet3p and the ferric permease Ftr1p. The research contained in this thesis describes the structural and functional features of Fet3p as they relate to its role in iron and copper homeostasis at the yeast plasma membrane. Chapter Two describes the identification of two previously uncharacterized iron binding ligands, D283 and D409, and examines their roles in substrate interactions, electron transfer processes, and Fe(III) trafficking to Ftr1p. Specifically, mutation of D283 or D409 to alanine resulted in ~4-fold decreases in Fe(II) affinity. Also, D409 acts as a viable electron transfer pathway by acting as a wire to guide electrons from Fe(II) to the T1 Cu site. During in vivo 59 Fe uptake assays both D283 and D409 mutants were sensitive to citrate concentration indicative of their involvement in ferric trafficking to Ftr1p. Chapter Three details a novel role for Fet3p in copper detoxification at the yeast plasma membrane. Yeast deficient in active Fet3p at the cell surface is sensitive to exogenous copper exposure. We have demonstrated that Fet3p and human ceruloplasmin exhibit high activity and high affinity for Cu(I) in addition to Fe(II). Mutation of Fet3p residue M345 to alanine results in ~2-fold increase in K M for Cu(I) and significantly reduced the rate of electron transfer from this substrate. Also, yeast expressing M345A exhibits an intermediary sensitivity to copper exposure. These results are striking evidence that Fet3p, as well as human ceruloplasmin are metallo-oxidases in that they can oxidize, at least, both iron and copper. Finally, Chapter Four focuses on the role of a conserved aspartate residue, D94, during enzyme turnover. D94 has been shown to act as an acid catalyst of oxygen reduction at the trinuclear cluster of an intermediate along the turnover cycle. Here we show that it also aids in the proton assisted rate determining step under turnover conditions.