Radical copper oxidases, one electron at a time

Whittaker, James W.; Whittaker, Mei M.

doi:10.1351/pac199870040903

Cited by 50 publications

(58 citation statements)

References 10 publications

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“…This displacement is coupled to a protonation event that implies that Tyr 495 can serve as a general base, capable of activating bound substrate by proton abstraction. A tryptophan residue (Trp 290 ) stacked over the thioether side chain (13) is thought to contribute to the unusual stability of the free radical-containing form of this enzyme, which can persist for weeks in the absence of reductants (20,21). Spectroscopic studies on the glyoxal oxidase (5) provide evidence for an active site structure nearly identical to that found in galactose oxidase, despite the distinct catalytic function.…”

Section: Glyoxal Oxidase (Glox)mentioning

confidence: 99%

Identification of Catalytic Residues in Glyoxal Oxidase by Targeted Mutagenesis

Whittaker¹,

Kersten²,

Cullen³

et al. 1999

Journal of Biological Chemistry

115

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Glyoxal oxidase is a copper metalloenzyme produced by the wood-rot fungus Phanerochaete chrysosporium as an essential component of its extracellular lignin degradation pathways. Previous spectroscopic studies on glyoxal oxidase have demonstrated that it contains a free radical-coupled copper active site remarkably similar to that found in another fungal metalloenzyme, galactose oxidase. Alignment of primary structures has allowed four catalytic residues of glyoxal oxidase to be targeted for site-directed mutagenesis in the recombinant protein. Three glyoxal oxidase mutants have been heterologously expressed in both a filamentous fungus (Aspergillus nidulans) and in a methylotrophic yeast (Pichia pastoris), the latter expression system producing as much as 2 g of protein per liter of culture medium under conditions of high density methanol-induced fermentation. Biochemical and spectroscopic characterization of the mutant enzymes supports structural correlations between galactose oxidase and glyoxal oxidase, clearly identifying the catalytically important residues in glyoxal oxidase and demonstrating the functions of each of these residues. Glyoxal oxidase (GLOX)1 from the wood-rot fungus Phanerochaete chrysosporium is a secreted enzyme that functions as an extracellular factory for production of hydrogen peroxide, fueling peroxidases (lignin peroxidase and manganese peroxidase) that are responsible for microbial lignin degradation (1-3). Glyoxal oxidase catalyzes the oxidization of aldehydes to carboxylic acids, coupled to reduction of dioxygen to hydrogen peroxide,The enzyme has fairly broad specificity for the reducing substrate, and a variety of simple dicarbonyl and hydroxycarbonyl compounds have been shown to support turnover. However, there is biological evidence that P. chrysosporium specifically secretes simple dicarbonyls (glyoxal and methylglyoxal) to drive this reaction. Further metabolism of the glyoxylic acid product leads to formation of oxalic acid, which has been identified as a cofactor for manganese peroxidase turnover (4). Previous studies of glyoxal oxidase (5) have demonstrated that it is a copper metalloenzyme containing an unusual free radical-coupled copper active site similar to that found in galactose oxidase (GAOX) (6, 7). In these enzymes, an amino acid side chain radical ligates the active site metal ion, forming a catalytic motif characteristic of a class of enzymes known as radical copper oxidases. This radical-copper complex acts as a two-electron redox active site, a distinction from other free radical enzymes (ribonucleotide reductase, etc.) (8 -12) which typically exhibit single-electron reactivity. Glyoxal oxidase is isolated as an inactive, reduced form lacking the free radical, and requires treatment with a strong oxidant (e.g. Ir(IV) or Mo(V)) for activation (5).The crystal structure of galactose oxidase (13) (Fig. 1) ) occur as consecutive residues in the protein sequence. The tyrosine ligands are distinct both in terms of coordination mode and, more especially, covalent m...

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Section: Glyoxal Oxidase (Glox)mentioning

confidence: 99%

Identification of Catalytic Residues in Glyoxal Oxidase by Targeted Mutagenesis

Whittaker¹,

Kersten²,

Cullen³

et al. 1999

Journal of Biological Chemistry

115

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“…Concurrently with the above work we prepared L 6 , which is a bidentate ligand containing an (Fig. 3) [31].…”

Section: Modeling the Tyrtrp π-π Interactionmentioning

confidence: 95%

“…Deconvolution of the voltammograms showed that the half-lives of 6 2+ and 7 2+ at 295 K were identical to each other within experimental error. Hence, the π-π interaction in 6.BF 4 has no bearing on the stability of the [L 6• ] + radical, beyond affording it some steric protection.…”

Section: Modeling the Tyrtrp π-π Interactionmentioning

confidence: 99%

“…+0.45 V vs. nHe, about half a volt less positive than for a "normal" tyrosine side-chain [5]. The resultant radical has a very unusual absorption spectrum, with an intense near-IR absorption between 600 and 1,200 nm, which has been suggested to arise from a Tyr → TyrCys • interligand chargetransfer (ILCT) process [6]. Once formed, the TyrCys • radical is very stable kinetically, with a half-life of up to a week under the correct conditions [6].…”

Section: Introductionmentioning

confidence: 99%

“…The resultant radical has a very unusual absorption spectrum, with an intense near-IR absorption between 600 and 1,200 nm, which has been suggested to arise from a Tyr → TyrCys • interligand chargetransfer (ILCT) process [6]. Once formed, the TyrCys • radical is very stable kinetically, with a half-life of up to a week under the correct conditions [6]. Alcohol oxidation by GOase proceeds by a free radical mechanism, for which abstraction of an H atom from the substrate by the TyrCys…”

Section: Introductionmentioning

confidence: 99%

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Dissecting an enzyme—model compounds for the galactose oxidase radical site

Halcrow

2002

Heteroatom Chemistry

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Ab initio molecular dynamics studies of a synthetic biomimetic model of galactose oxidase

Röthlisberger

Carloni

1999

Int. J. Quant. Chem.

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Very recently, highly efficient biomimetic models of the mononuclear copper enzyme galactose oxidase were synthesized which are able to reproduce the structural, spectroscopic, and functional properties of the native system exceptionally well. We have characterized an inactive and an active form of one of these biomimetic compounds using unrestricted dynamical density functional calculations. The peculiar nonsquare planar O N -coordination geometry of the copper ion in the catalytically 2 2 Ž . inactive EPR-active form induces a complex energy-level diagram that cannot be related to crystal-field models: The highest occupied orbitals are located on the -system of the aromatic ligands and are essentially spin-paired while the unpaired electron is localized mainly in a lower-lying d 2 2 orbital of the copper. Using ab initio molecular x yy dynamics simulations, we determined for the first time the structure of the active form complexed with a substrate analog. Our calculations reveal that upon substrate binding one of the phenolate ligands is pushed away from the copper center into an axial position and the electronic structure rearranges to an unusual antiferromagnetic diradical state. As in the inactive form, the unpaired ␣-spin density is located in the copper d 2 2x yy orbital. The unpaired ␤-spin density, instead, is localized on the axial ligand in agreement with the ligand-based radical mechanism that has been proposed for galactose oxidase.

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Radical copper oxidases, one electron at a time

Cited by 50 publications

References 10 publications

Identification of Catalytic Residues in Glyoxal Oxidase by Targeted Mutagenesis

Identification of Catalytic Residues in Glyoxal Oxidase by Targeted Mutagenesis

Dissecting an enzyme—model compounds for the galactose oxidase radical site

Ab initio molecular dynamics studies of a synthetic biomimetic model of galactose oxidase

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