School of Chemistry - Research Publications

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    Glutaredoxins employ parallel monothiol-dithiol mechanisms to catalyze thiol-disulfide exchanges with protein disulfides
    Ukuwela, AA ; Bush, AI ; Wedd, AG ; Xiao, Z (ROYAL SOC CHEMISTRY, 2018-02-07)
    Glutaredoxins (Grxs) are a family of glutathione (GSH)-dependent thiol-disulfide oxidoreductases. They feature GSH-binding sites that directly connect the reversible redox chemistry of protein thiols to the abundant cellular nonprotein thiol pool GSSG/GSH. This work studied the pathways for oxidation of protein dithiols P(SH)2 and reduction of protein disulfides P(SS) catalyzed by Homo sapiens HsGrx1 and Escherichia coli EcGrx1. The metal-binding domain HMA4n(SH)2 was chosen as substrate as it contains a solvent-exposed CysCys motif. Quenching of the reactions with excess iodoacetamide followed by protein speciation analysis via ESI-MS allowed interception and characterization of both substrate and enzyme intermediates. The enzymes shuttle between three catalytically-competent forms (Grx(SH)(S-), Grx(SH)(SSG) and Grx(SS)) and employ conserved parallel monothiol and dithiol mechanisms. Experiments with dithiol and monothiol versions of both Grx enzymes demonstrate which monothiol (plus GSSG or GSH) or dithiol pathways dominate a specific oxidation or reduction reaction. Grxs are shown to be a class of versatile enzymes with diverse catalytic functions that are driven by specific interactions with GSSG/GSH.
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    The challenges of using a copper fluorescent sensor (CS1) to track intracellular distributions of copper in neuronal and glial cells
    Price, KA ; Hickey, JL ; Xiao, Z ; Wedd, AG ; James, SA ; Liddell, JR ; Crouch, PJ ; White, AR ; Donnelly, PS (ROYAL SOC CHEMISTRY, 2012)
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    Unification of the Copper(I) Binding Affinities of the Metallo-chaperones Atx1, Atox1, and Related Proteins DETECTION PROBES AND AFFINITY STANDARDS
    Xiao, Z ; Brose, J ; Schimo, S ; Ackland, SM ; La Fontaine, S ; Wedd, AG (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011-04-01)
    Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. In this study, application of the four Cu(I) ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their Cu(I) affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with Cu(I) to yield distinct 1:2 chromatophoric complexes [Cu(I)L(2)](3-) with formation constants β(2) = 10(17.2) and 10(19.8) m(-2), respectively. These constants do not depend on proton concentration for pH ≥7.0. Consequently, they are a pair of complementary and stable probes capable of detecting free Cu(+) concentrations from 10(-12) to 10(-19) m. Dtt binds Cu(I) with K(D) ∼10(-15) m at pH 7, but it is air-sensitive, and its Cu(I) affinity varies with pH. The Cu(I) binding properties of Atox1 and related proteins (including the fifth and sixth domains at the N terminus of the Wilson protein ATP7B) were assessed with these probes. The results demonstrate the following: (i) their use permits the stoichiometry of high affinity Cu(I) binding and the individual quantitative affinities (K(D) values) to be determined reliably via noncompetitive and competitive reactions, respectively; (ii) the scattered literature values are unified by using reliable probes on a unified scale; and (iii) Atox1-type proteins bind Cu(I) with sub-femtomolar affinities, consistent with tight control of labile Cu(+) concentrations in living cells.
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    Metallo-oxidase Enzymes: Design of their Active Sites
    Xiao, Z ; Wedd, AG (CSIRO PUBLISHING, 2011)
    Multi-copper oxidases are a large family of enzymes prevalent in all three domains of life. They couple the one-electron oxidation of substrate to the four-electron reduction of dioxygen to water and feature at least four Cu atoms, traditionally divided into three sites: T1, T2, and (binuclear) T3. The T1 site catalyzes substrate oxidation while a trinuclear cluster (comprising combined T2 and T3 centres) catalyzes the reduction of dioxygen. Substrate oxidation at the T1 Cu site occurs via an outer-sphere mechanism and consequently substrate specificities are determined primarily by the nature of a substrate docking/oxidation (SDO) site associated with the T1 Cu centre. Many of these enzymes ‘moonlight’, i.e. display broad specificities towards many different substrates and may have multiple cellular functions. A sub-set are robust catalysts for the oxidation of low-valent transition metal ions such as FeII, CuI, and MnII and are termed ‘metallo-oxidases’. They play essential roles in nutrient metal uptake and homeostasis, with the ferroxidase ceruloplasmin being a prominent member. Their SDO sites are tailored to facilitate specific binding and facile oxidation of these low-valent metal ions and this is the focus of this review.