Defining the interplay between copper transfer and disulfide oxidation in copper zinc superoxide dismutase

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Title Defining the interplay between copper transfer and disulfide oxidation in copper zinc superoxide dismutase
Publication Type dissertation
School or College School of Medicine
Department Biochemistry
Author Fetherolf, Morgan Minoru
Date 2017
Description Copper zinc superoxide dismutase (Sod1) is a critical enzyme in limiting reactive oxygen species in both the cytosol and the mitochondrial inner membrane space. Sod1 dismutes superoxide anions to hydrogen peroxide and oxygen. The catalytic reaction is dependent on an active site copper ion and a disulfide-bonded conformation. The copper chaperone for superoxide dismutase (Ccs1) mediates the activation of Sod1 either through facilitated copper ion loading, disulfide bond formation or both. In the past several decades, Sod1 has been studied extensively due to frequent mutations found in the familial form of amyotrophic lateral sclerosis (ALS). To date, over 150 mutations have been characterized in Sod1; however, the basic maturation process of the enzyme is poorly understood. In this study, we explore the basic mechanism by which Sod1 matures. To define the activation process, we have performed extensive mutagenesis on the Cys residues in Ccs1. These mutations show a strict dependence on the CXC motif in domain 3, and the spacing of the two Cys residues. Mutations that alter the spacing or remove one of the cysteinyl residues from the CXC motif result in defects in copper metallation and disulfide oxidation. Mutations of the second Cys in the Ccs1 domain 3 or Cys146 of Sod1, which participates in the intramolecular disulfide, results in enhanced stalling of the heterdimeric complex between Ccs1 and Sod1 when affinity purification of Sod1 was performed. The two Sod1 cysteinyl residues exhibit differential phenotypes in copper loading. A C57S Sod1 mutant is catalytically dead and devoid of bound copper when purified from yeast. In contrast, a C146S mutant is partially copper loaded and exhibits weak Sod1 activity. C57S is proposed to serve as an entry site ligand during Cu(I) loading. In an attempt to observe an entry site Cu(I) site, X-ray absorption spectroscopy was performed on Sod1 and Ccs1 mutants that stall the heterodimer. The Cu(I) coordination site with thiolate ligands was observed. We believe that this entry site is functional, and that a single redox turnover of Cu(I) to Cu(II) is necessary to generate hydrogen peroxide. We characterize that through a reaction of hydrogen peroxide with a free thiol, cysteine146 of Sod1, a sulfenic acid intermediate is fomed. We propose that the sulfenic acid intermediate is able to undergo a nucleophilic attack, potential from a thiolate from the CXC motif in Ccs1, to promote a disulfide exchange reaction.
Type Text
Publisher University of Utah
Subject copper; superoxide; superoxide dismutase
Dissertation Name Doctor of Philosophy
Language eng
Rights Management ©Morgan Minoru Fetherolf
Format application/pdf
Format Medium application/pdf
ARK ark:/87278/s62g1snc
Setname ir_etd
ID 1345119
Reference URL https://collections.lib.utah.edu/ark:/87278/s62g1snc
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