Spectroscopic Analysis of the Trinuclear Cluster in the Fet3 Protein from Yeast, a Multinuclear Copper Oxidase

Blackburn, Ninian J.; Ralle, Martina; Hassett, Richard; Kosman, Daniel J.

doi:10.1021/bi992334a

Cited by 67 publications

(95 citation statements)

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“…In Fet3pT1D, both T3 and T2 sites are present, completing the TNC at the interface of domains 1 and 3. Again, spectral evidence on this variant shows this cluster to have native-like electronic properties (17). We find that addition of T2 to T3-containing Fet3p increases overall protein stability by Ϸ5°C (T m of 55°C for Fet3pT1D; 1 K/min) but the thermal process remains coupled in a single step (Fig.…”

Section: Resultsmentioning

confidence: 66%

“…In this protein, the binuclear T3 cluster at the interface is present, but not T1 and T2. Absorbance and extended x-ray absorption fine structure spectra of Fet3pT1D/T2D indicate that T3 is native-like in its electronic characteristics (6,17). In Fig.…”

Section: Resultsmentioning

confidence: 88%

“…WT (with and without carbohydrates) and mutant Fet3p variants was prepared as described (17,32). The variants used are as follows: T1D contains a Cys-484-to-Ser mutation at the T1 site; T2D includes a His-81-to-Gln mutation at the T2 site; T1D/T2D is a double mutant with both the above changes introduced (17).…”

Section: Methodsmentioning

confidence: 99%

“…Yeast ferroxidase, Fet3p, has proven to be a paradigm for structure-function studies because of the collection of demetallated forms that have allowed quantification of the role of each Cu site to the electronic properties of Fet3p and to its redox activity (6,(17)(18)(19). These forms are designated T1D, T2D, and T1D/T2D (where D is depleted), in which the T1, T2, or both the T1 and T2 Cu atoms are absent because of mutation of specific Cu ligands.…”

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confidence: 99%

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In vitro unfolding of yeast multicopper oxidase Fet3p variants reveals unique role of each metal site

Sedlák

Ziegler

Kosman

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Fet3p from Saccharomyces cerevisiae is a multicopper oxidase (MCO) that contains 3 cupredoxin-like ␤-barrel domains and 4 copper ions located in 3 distinct metal sites (T1 in domain 3, T2, and the binuclear T3 at the interface between domains 1 and 3). To better understand how protein structure and stability is defined by cofactor coordination in MCO proteins, we assessed thermal unfolding of apo and metallated forms of Fet3p by using spectroscopic and calorimetric methods in vitro (pH 7). We find that unfolding reactions of apo and different holo forms of Fet3p are irreversible reactions that depend on the scan rate. The domains in apo-Fet3p unfold sequentially [thermal midpoint (T m) of 45°C, 62°C, and 72°C; 1 K/min]. Addition of T3 imposes strain in the apo structure that results in coupled domain unfolding and low stability (T m of 50°C; 1 K/min). Further inclusion of T2 (i.e., only T1 absent) increases overall stability by Ϸ5°C but unfolding remains coupled in 1 step. Introduction of T1, producing fully-loaded holo-Fet3p (or in the absence of T2), results in stabilization of domain 3, which uncouples unfolding of the domains; unfolding of domain 2 occurs first along with Cu-site perturbations (T m 50 -55°C; 1 K/min), followed by unfolding of domains 1 and 3 (Ϸ65-70°C; 1 K/min). Our results suggest that there is a metal-induced tradeoff between overall protein stability and metal coordination in members of the MCO family.spectroscopy ͉ calorimetry ͉ cupredoxin ͉ copper-binding protein

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Section: Resultsmentioning

confidence: 66%

Section: Resultsmentioning

confidence: 88%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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In vitro unfolding of yeast multicopper oxidase Fet3p variants reveals unique role of each metal site

Sedlák

Ziegler

Kosman

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…27,28 (Scheme 1, top) Derivatives of the native enzyme have been prepared where the T1 is either eliminated 29,30 (replacement of the Cys ligand of the T1 with a Ser to generate a type 1 depleted or T1D form) or replaced by a redox innocent mercuric ion (the T1Hg derivative). 31,32 These have valid TNCs which, when reduced, react with O 2 with essentially the same rate constant as the native enzyme.…”

Section: O 2 Reactivity: the Trinuclear Cu Cluster (Tnc)mentioning

confidence: 99%

O2 Reduction to H2O by the multicopper oxidases

2008

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In nature the four electron reduction of O 2 to H 2 O is carried out by Cytochrome c Oxidase (CcO) and the multicopper oxidases (MCOs). In the former, Cytochrome c provides electrons for pumping protons to produce a gradient for ATP synthesis, while in the MCOs the function is the oxidation of substrates, either organic or metal ions. In the MCOs the reduction of O 2 is carried out at a trinuclear Cu cluster (TNC). Oxygen intermediates have been trapped which exhibit unique spectroscopic features that reflect novel geometric and electronic structures. These intermediates have both intact and cleaved O-O bonds, allowing the reductive cleavage of the O-O bond to be studied in detail both experimentally and computationally. These studies show that the topology of the TNC provides a unique geometric and electronic structure particularly suited to carry out this key reaction in Nature.The multicopper oxidases (MCOs) couple four 1-electron oxidations of substrate to the four electron reductive cleavage of the O-O bond of dioxygen using a minimum of four Cu atoms (table 1). 1,2 Among these four Cu's is a type 1 (T1) or blue Cu site, characterized by an intense S Cys → Cu(II) charge transfer (CT) transition at around 600 nm in the absorption spectrum and a uniquely small A || in its electron paramagnetic resonance (EPR) spectrum. This is the site of substrate oxidation, and from table 1, the MCOs can be divided into two classes depending upon the identity of the substrate. For enzymes such as laccase 3 and ascorbate oxidase, 4 redox active organic molecules which can interact weakly with the enzyme provide the electrons. For MCOs like Fet3p 5 and Ceruloplasmin, 6 the substrate is a metal ion (ferrous in these cases) which binds tightly to a substrate binding site. As shown in figure 1, these substrate binding sites are located near the His ligands of the T1 Cu center. The electron from substrate is first transferred to the T1 and then over >13Å through a Cys-His pathway to a trinuclear Cu cluster (TNC) where O 2 is reduced to water (vide infra). 7 We first consider the electron transfer (ET) pathways to the TNC. ET PathwaysHere we focus on the Fe(II) binding site of the enzyme Fet3p, which is involved in the uptake of iron by yeast. 8 (Studies on this enzyme were performed in collaboration with Prof. Dan Kosman and coworkers.) A variable-temperature, variable-field magnetic circular dichroism (VTVH MCD) methodology we developed in other studies was applied to probe this ferrous site. [9][10][11] From figure 2A dark blue, there is a characteristic feature at 8900 cm −1 in the MCD spectrum corresponding to Fe(II) binding with a high affinity (K B > 10 5 M −1 , from MCD titration studies) to a 6 coordinate site in the protein. 12 In the light blue spectrum this feature is eliminated and a peak at 9700 cm −1 , corresponding to aqueous Fe(II) (green) is observed when Zn(II) is first bound to the substrate site, inhibiting ferroxidase activity.From mutagenesis studies we have found that three carboxylates are involved...

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Copper Proteins: Oxidases

Stoj¹,

Kosman²

2005

Encyclopedia of Inorganic Chemistry

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Oxidases are enzymes that use dioxygen as the electron acceptor in oxido‐reduction reactions. Many members of this enzyme class (EC 1.) rely on the Cu 1+ /Cu 2+ redox cycle of copper prosthetic group(s) in their catalysis of the electron transfer from reducing substrate to O 2 . Those copper oxidases that catalyze the four‐electron reduction of O 2 to 2H 2 O are known as multicopper oxidases (MCO) because each member of this group contains at least four copper atoms of three distinct electronic and spectral types. These three types are: type 1 or ‘blue’ Cu(II), so‐called because of its distinguishing strong absorbance at ∼600 nm; type 2 Cu(II), comparable in electronic structure to that found in square‐planar copper complexes; and type 3 Cu(II), a Cu(II)Cu(II) pair bridged by O(H) and thus diamagnetic. With four 1e − metal redox sites, MCOs are the all‐copper equivalent of the copper‐heme respiratory terminal oxidases. However, MCOs are true oxidases in that all members can use as electron acceptors common (di)phenolic, and aromatic (di)amine substrates, for example, hydroquinone, ascorbic acid, and phenylenediamine. The laccases and ascorbate oxidase are examples of these MCOs (EC 1.10.3). Members of a separate MCO class (EC 1.16.3) also use lower valent metal ions as substrates, for example, Fe 2+ , Cu 1+ , and/or Mn 2+ . These are thereby designated metallo‐oxidases; the most widely studied activity of this class has been towards Fe 2+ giving rise to the common enzyme name, ferroxidase. The typical MCO structure consists of three ‘cuprodoxin’ domains, a Greek‐key beta‐fold that characterizes a family of mononuclear copper electron‐transfer factors. Among MCOs are both soluble and type 1 membrane proteins. Functionally, MCOs are known to play essential roles in biodegradation and transformation, cell redox balance, and metal metabolism. This article summarizes current knowledge of this class of copper oxidase enzymes.

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Spectroscopic Analysis of the Trinuclear Cluster in the Fet3 Protein from Yeast, a Multinuclear Copper Oxidase

Cited by 67 publications

References 41 publications

In vitro unfolding of yeast multicopper oxidase Fet3p variants reveals unique role of each metal site

In vitro unfolding of yeast multicopper oxidase Fet3p variants reveals unique role of each metal site

O2 Reduction to H2O by the multicopper oxidases

Copper Proteins: Oxidases

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