<scp>l</scp>-Tryptophan Radical Cation Electron Spin Resonance Studies: Connecting Solution-Derived Hyperfine Coupling Constants with Protein Spectral Interpretations

Connor, Henry D.; Sturgeon, Bradley E.; Mottley, Carolyn; Sipe, Herbert J.; Mason, Ronald P.

doi:10.1021/ja0780277

Cited by 35 publications

(24 citation statements)

References 38 publications

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“…A similar g-tensor anisotropy effect would be expected for a hydroxylated Trp radical because the radical spin density is typically delocalized over the entire indole ring (22)(23)(24)(25)(26)(27). Therefore, βTrp57-OH of preMADH would be expected to have a Δg value greater than that of Trp radicals and closer to that of Tyr radicals.…”

Section: Resultsmentioning

confidence: 76%

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Yukl

Liu

Krzystek

et al. 2013

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

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Despite the importance of tryptophan (Trp) radicals in biology, very few radicals have been trapped and characterized in a physiologically meaningful context. Here we demonstrate that the diheme enzyme MauG uses Trp radical chemistry to catalyze formation of a Trp-derived tryptophan tryptophylquinone cofactor on its substrate protein, premethylamine dehydrogenase. The unusual sixelectron oxidation that results in tryptophan tryptophylquinone formation occurs in three discrete two-electron catalytic steps. Here the exact order of these oxidation steps in the processive six-electron biosynthetic reaction is determined, and reaction intermediates are structurally characterized. The intermediates observed in crystal structures are also verified in solution using mass spectrometry. Furthermore, an unprecedented Trp-derived diradical species on premethylamine dehydrogenase, which is an intermediate in the first two-electron step, is characterized using high-frequency and -field electron paramagnetic resonance spectroscopy and UV-visible absorbance spectroscopy. This work defines a unique mechanism for radical-mediated catalysis of a protein substrate, and has broad implications in the areas of applied biocatalysis and understanding of oxidative protein modification during oxidative stress.cofactor biosynthesis | tryptophan radical | heme | posttranslational modification | electron transfer P rotein-based radicals, particularly on tyrosine (Tyr) and tryptophan (Trp) residues, have been implicated in a large number of catalytic and electron transfer reactions in biology (1), including the long-range electron transfer reactions required for photosynthesis (2), respiration (3), and DNA synthesis (4) and repair (5). Aberrant formation of protein radicals during oxidative stress is also of special importance. Evidence for the involvement of protein-based and substrate-based radicals in enzyme-catalyzed reactions has increased substantially in recent years, and expanded our knowledge of the scope of chemical reactions accessible to enzymes. The ability of enzymes to catalyze what were previously thought to be unattainable reactions is exemplified by MauG. The biosynthesis of the tryptophan tryptophylquinone (TTQ) cofactor in methylamine dehydrogenase (MADH) requires posttranslational modification of two tryptophan residues. MADH from Paracoccus denitrificans is a 119-kDa α 2 β 2 heterotetramer that contains two active sites and two TTQ cofactors derived from residues βTrp57 and βTrp108 (6). MauG is a c-type diheme enzyme that catalyzes the conversion of a MADH precursor (preMADH) that has one oxygen atom already inserted into the βTrp57 indole ring (βTrp57-OH of preTTQ) (7), to mature TTQ-containing MADH (8) (Fig. 1).Catalysis by MauG proceeds via a bis-Fe(IV) redox state, which may be generated by reaction of di-Fe(II) MauG with O 2 or di-Fe(III) MauG with H 2 O 2 (9). Catalytically active crystals of the MauG-preMADH protein complex show that the site of TTQ formation on β-preMADH is 40.1 Å from the MauG highspin heme iron...

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Section: Resultsmentioning

confidence: 76%

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Yukl

Liu

Krzystek

et al. 2013

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

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“…6(b)), replacing the suggested hfcc of the hydrogen atom of the NH group with the deuterium parameters ( Table 2). [35] Possible reaction routes in anodic oxidation EPR data brought satisfactory evidence that the radical observed in 0.1 M NaOH originates from N(1)-deprotonated, and in 0.001 M from N(1)-protonated oxo-tautomer of 4-oxoquinoline semiquinone radical anions. The reaction system employed in the EPR study is relatively complex.…”

Section: Anodic Oxidation In 0001 M Naoh (Ph ∼ 11)mentioning

confidence: 90%

Anodic oxidation of selenadiazoloquinolones in alkaline media

Staško

Zalibera

Barbieriková

et al. 2011

Magnetic Reson in Chemistry

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Newly synthesized derivatives of 6-oxo-6,9-dihydro[1,2,5]selenadiazolo[3,4-h]quinoline variously substituted at position 7 (R = H, COOH, COCH(3), CN, COOC(2)H(5) and COOCH(3)) are established in strongly alkaline aqueous solutions (0.1 M NaOH; pH ∼ 13) as N(9)-deprotonated structures, but in less alkaline solutions (0.001 M NaOH; pH ∼ 11) the N(9)-protonated oxo tautomeric forms dominate. Upon their anodic oxidation in alkaline solutions, the selenadiazole ring is replaced, forming instead the paramagnetic species analogous to the ortho semiquinone radical anions as monitored by in situ EPR spectroscopy. The quantum chemical calculations for two representative selenadiazoloquinolones (R = H and COOH) and their anodic oxidation products presented are in agreement with experiments.

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“…Direct ESR is the gold standard for detection of free radicals, and is most useful for studies of relatively stable radicals. Connor et al [20], for example, published the first report of an L-tryptophan radical cation detected using fast-flow ESR and acidic Ce 4+ as the oxidizing agent. Detection of tryptophan radical in proteins, however, is complicated by the ability of other amino acid residues to form radicals, and the possibility that radical transfer could occur between residues within a single molecule [21, 22] or between residues of two individual molecules [2].…”

Section: Detection Methodsmentioning

confidence: 99%

Tripping up Trp: Modification of protein tryptophan residues by reactive oxygen species, modes of detection, and biological consequences

Ehrenshaft

Deterding

Mason

2015

Free Radical Biology and Medicine

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121

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Proteins comprise a majority of the dry weight of a cell, rendering them a major target for oxidative modification. Oxidation of proteins can result in significant alterations in protein molecular mass such as breakage of the polypeptide backbone, and/or polymerization of monomers into dimers, multimers and sometimes into insoluble aggregates. Protein oxidation can also result in structural changes to amino acid residue side chains, conversions which have only a modest effect on protein size but can have widespread consequences for protein function. There are a wide range of rate constants for amino acid reactivity, with cysteine, methionine, tyrosine, phenylalanine and tryptophan having the highest rate constants with commonly encountered biological oxidants. Free tryptophan and tryptophan protein residues react at a diffusion limited rate with hydroxyl radical, and also have high rate constants for reactions with singlet oxygen and ozone. Although oxidation of proteins in general and tryptophan residues specifically can have effects detrimental to the health of cells and organisms, some modifications are neutral while others contribute to the function of the protein in question or may act as a signal that damaged proteins need to be replaced. This review provides a brief overview of the chemical mechanisms by which tryptophan residues become oxidized, presents both the strengths and weaknesses of some of the techniques used to detect these oxidative interactions and discusses selected examples of the biological consequences of tryptophan oxidation in proteins from animals, plants and microbes.

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l-Tryptophan Radical Cation Electron Spin Resonance Studies: Connecting Solution-Derived Hyperfine Coupling Constants with Protein Spectral Interpretations

Cited by 35 publications

References 38 publications

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Anodic oxidation of selenadiazoloquinolones in alkaline media

Tripping up Trp: Modification of protein tryptophan residues by reactive oxygen species, modes of detection, and biological consequences

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