Dioxygen Inactivation of Pyruvate Formate-Lyase:  EPR Evidence for the Formation of Protein-Based Sulfinyl and Peroxyl Radicals

Reddy, Sreelatha G.; Wong, Kenny K.; Parast, Camran V.; Peisach, Jack; Magliozzo, Richard S.; Kozarich, John W.

doi:10.1021/bi972086n

Cited by 70 publications

(98 citation statements)

References 24 publications

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“…First, the lineshapes at low or higher temperatures are inconsistent with other radicals that have been observed in proteins, such as glycyl (43), cysteinyl (44), or tryptophanyl (45,46). Some similarity exists between the spectral envelope of the present species and that of a sulfinyl radical observed during oxygen-dependent inactivation of pyruvate-formate lyase (47), and that seen in the Y177F/ I263C double mutant of murine type 1 ribonucleotide reductase R2 protein (48); however, three pieces of evidence argue against this possibility in the case of OxDC. First, sulfinyl radicals reported show a relatively large g x value of ϳ2.02; our spectra cannot be simulated with a value this large.…”

Section: Nature Of the Manganese Center In Oxalate Decarboxylase-contrasting

confidence: 57%

EPR Spectroscopic Characterization of the Manganese Center and a Free Radical in the Oxalate Decarboxylase Reaction

Chang

Svedružić

Ożarowski

et al. 2004

Journal of Biological Chemistry

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Several molecular mechanisms for cleavage of the oxalate carbon-carbon bond by manganese-dependent oxalate decarboxylase have recently been proposed involving high oxidation states of manganese. We have examined the oxalate decarboxylase from Bacillus subtilis by electron paramagnetic resonance in perpendicular and parallel polarization configurations to test for the presence of such species in the resting state and during enzymatic turnover. Simulation and the position of the half-field Mn(II) line suggest a nearly octahedral metal geometry in the resting state. No spectroscopic signature for Mn(III) or Mn(IV) is seen in parallel mode EPR for samples frozen during turnover, consistent either with a large zero-field splitting in the oxidized metal center or undetectable levels of these putative high-valent intermediates in the steady state. A narrow, featureless g ‫؍‬ 2.0 species was also observed in perpendicular mode in the presence of substrate, enzyme, and dioxygen. Additional splittings in the signal envelope became apparent when spectra were taken at higher temperatures. Isotopic editing resulted in an altered line shape only when tyrosine residues of the enzyme were specifically deuterated. Spectral processing confirmed multiple splittings with isotopically neutral enzyme that collapsed to a single prominent splitting in the deuterated enzyme. These results are consistent with formation of an enzyme-based tyrosyl radical upon oxalate exposure. Modestly enhanced relaxation relative to abiological tyrosyl radicals was observed, but site-directed mutagenesis indicated that conserved tyrosine residues in the active site do not host the unpaired spin. Potential roles for manganese and a peripheral tyrosyl radical during steady-state turnover are discussed.The oxalate decarboxylase (OxDC, 1 or YvrK based on the corresponding open reading frame) from Bacillus subtilis has recently garnered attention because of its mechanistically intriguing reaction (1-4) (Scheme 1). Purified OxDC crystallizes as a hexamer of 43-kDa subunits, each of which is a member of the "bicupin" structural family (5), and contains two mononuclear manganese centers with amino acid ligands that are functionally similar to those of manganese superoxide dismutase (MnSOD) (6); namely three histidine residues and one carboxylic acid residue. Oxalate 13 C and 18 O isotope effects on V max /K m of YvrK suggest the presence of a slow, partially rate-determining step prior to the irreversible C-C bond cleavage (4), which was proposed to be a proton-coupled electron transfer.The requirement for and substoichiometric consumption of dioxygen in the YvrK catalytic process, involvement of open shell manganese (2), and chemical precedent for oxalate decarboxylation in the Kolbe electrolysis (7,8) and the BorodinHunsdiecker reaction (9 -11) have led to various mechanistic proposals involving radical intermediates, and enzymic manganese complexes with formal oxidation states of either Mn(III) (2, 4) or Mn(IV) (3). A previously published electron paramagneti...

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Section: Nature Of the Manganese Center In Oxalate Decarboxylase-contrasting

confidence: 57%

EPR Spectroscopic Characterization of the Manganese Center and a Free Radical in the Oxalate Decarboxylase Reaction

Chang

Svedružić

Ożarowski

et al. 2004

Journal of Biological Chemistry

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“…The PFL/O 2 samples employed still contained about 25% native polypeptide chains (␣, 85 kDa) and could therefore yield a small recovery of PFL activity without addition of YfiD or Y06I. Of the fragmented polypeptide chains (␤, 82 kDa) contained in PFL/O 2 , a major fraction was presumably oxidized ir-reversibly at Cys419, as was previously documented for the oxygen-reaction of PFL enzyme studied in vitro (17). This may explain the incomplete restoration of catalytic activity in the presence of the helper proteins (about 40 U/mg compared to 200 U/mg for native PFL).…”

Section: Resultsmentioning

confidence: 59%

YfiD of Escherichia coli and Y06I of Bacteriophage T4 as Autonomous Glycyl Radical Cofactors Reconstituting the Catalytic Center of Oxygen-Fragmented Pyruvate Formate-Lyase

Schultz

Bomke

et al. 2001

Biochemical and Biophysical Research Communications

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“…In pyruvate formate lyase a disulfide radical is formed when the enzyme is inactivated with mercaptopyruvate, and a sulfinyl (and a peroxyl) radical is formed when pyruvate formate lyase is inactivated in the presence of oxygen (21,22). The disulfide radical has g x ϭ 2.057, g y ϭ 2.023, g z Ϸ 2.00, and the sulfinyl radical has g x ϭ 2.0204, g y ϭ 2.0084, g z ϭ 2.0005.…”

Section: Discussionmentioning

confidence: 99%

Cysteinyl and Substrate Radical Formation in Active Site Mutant E441Q of Escherichia coli Class I Ribonucleotide Reductase

Persson

Sahlin

Sjöberg

1998

Journal of Biological Chemistry

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All classes of ribonucleotide reductase are proposed to have a common reaction mechanism involving a transient cysteine thiyl radical that initiates catalysis by abstracting the 3-hydrogen atom of the substrate nucleotide. In the class Ia ribonucleotide reductase system of Escherichia coli, we recently trapped two kinetically coupled transient radicals in a reaction involving the engineered E441Q R1 protein, wild-type R2 protein, and substrate (Persson, A. L., Eriksson, M., Katterle, B., Pö tsch, S., Sahlin, M., and Sjö berg, B.-M. (1997) J. Biol. Chem. 272, 31533-31541). Using isotopically labeled R1 protein or substrate, we now demonstrate that the early radical intermediate is a cysteinyl radical, possibly in weak magnetic interaction with the diiron site of protein R2, and that the second radical intermediate is a carbon-centered substrate radical with hyperfine coupling to two almost identical protons. This is the first report of a cysteinyl free radical in ribonucleotide reductase that is a kinetically coupled precursor of an identified substrate radical. We suggest that the cysteinyl radical is localized to the active site residue, Cys 439 , which is conserved in all classes of ribonucleotide reductase, and which, in the three-dimensional structure of protein R1, is positioned to abstract the 3-hydrogen atom of the substrate. We also suggest that the substrate radical is localized to the 3-position of the ribose moiety, the first substrate radical intermediate in the postulated reaction mechanism.De novo synthesis of deoxyribonucleotides from ribonucleotides is catalyzed by the enzyme ribonucleotide reductase (RNR).1 All currently known classes of RNR are considered to have a common reaction mechanism based on radical chemistry (1). The RNR classes have different subunit compositions and different metal/radical cofactor requirements, but recent structural data suggest that their substrate binding domains are homologous.2 The proposed reaction mechanism includes formation of a transient thiyl radical at the active site, which, by abstracting the 3Ј-hydrogen atom of the substrate, generates a transient 3Ј substrate radical (1, 2). The formation of this 3Ј nucleotide radical may be the critical step (3) for the subsequent steps of the reaction mechanism.The aerobic class Ia RNR of Escherichia coli, is composed of two homodimeric components, protein R1 and protein R2, of known three-dimensional structures (4, 5). Protein R1 is the substrate binding component and Cys 439 at the active site is postulated to harbor the transient thiyl radical. The cysteinyl radical is thought to be generated by long range radical transfer to the R2 component, which harbors a stable oxidized tyrosyl radical in close proximity to a diiron-oxo site. The radical transfer presumably occurs via a specific route involving at least 6 residues in R2 ( ). These residues have been conserved in class I RNRs (1), which are found in all studied eukaryotes (except Euglena), and in some eubacteria and archea (6).The in vitro k cat for the aerob...

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Dioxygen Inactivation of Pyruvate Formate-Lyase: EPR Evidence for the Formation of Protein-Based Sulfinyl and Peroxyl Radicals

Cited by 70 publications

References 24 publications

EPR Spectroscopic Characterization of the Manganese Center and a Free Radical in the Oxalate Decarboxylase Reaction

EPR Spectroscopic Characterization of the Manganese Center and a Free Radical in the Oxalate Decarboxylase Reaction

YfiD of Escherichia coli and Y06I of Bacteriophage T4 as Autonomous Glycyl Radical Cofactors Reconstituting the Catalytic Center of Oxygen-Fragmented Pyruvate Formate-Lyase

Cysteinyl and Substrate Radical Formation in Active Site Mutant E441Q of Escherichia coli Class I Ribonucleotide Reductase

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