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Query: UNIPROT:O14944 (EPR)
 13,097 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The last step of the biosynthesis of fosfomycin, a clinically useful antibiotic, is the conversion of (S)-2-hydroxypropylphosphonic acid (HPP) to fosfomycin. Since the ring oxygen in fosfomycin has been shown in earlier feeding experiments to be derived from the hydroxyl group of HPP, this oxirane formation reaction is effectively a dehydrogenation process. To study this unique C-O bond formation step, we have overexpressed and purified the desired HPP epoxidase. Results reported herein provided initial biochemical evidence revealing that HPP epoxidase is an iron-dependent enzyme and that both NAD(P)H and a flavin or flavoprotein reductase are required for its activity. The 2 K EPR spectrum of oxidized iron-reconstituted fosfomycin epoxidase reveals resonances typical of S = (5)/(2) Fe(III) centers in at least two environments. Addition of HPP causes a redistribution with the appearance of at least two additional species, showing that the iron environment is perturbed. Exposure of this sample to NO elicits no changes, showing that the iron is nearly all in the Fe(III) state. However, addition of NO to the Fe(II) reconstituted enzyme that has not been exposed to O(2) yields an intense EPR spectrum typical of an S = (3)/(2) Fe(II)-NO complex. This complex is also heterogeneous, but addition of substrate converts it to a single, homogeneous S = (3)/(2) species with a new EPR spectrum, suggesting that substrate binds to or near the iron, thereby organizing the center. The fact that NO binds to the ferrous center suggests O(2) can also bind at this site as part of the catalytic cycle. Using purified epoxidase and (18)O isotopic labeled HPP, the retention of the hydroxyl oxygen of HPP in fosfomycin was demonstrated. While ether ring formation as a result of dehydrogenation of a secondary alcohol has precedence in the literature, these catalyses require alpha-ketoglutarate for activity. In contrast, HPP epoxidase is alpha-ketoglutarate independent. Thus, the cyclization of HPP to fosfomycin clearly represents an intriguing conversion beyond the scope entailed by common biological epoxidation and C-O bond formation.
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PMID:Biochemical and spectroscopic studies on (S)-2-hydroxypropylphosphonic acid epoxidase: a novel mononuclear non-heme iron enzyme. 1452 67

Insertion mutant Ins2 of the cyanobacterium Synechocystis sp. PCC 6803, lacking NAD(P)H:quinone oxidoreductase (NQR) encoded by drgA gene, was characterized by higher sensitivity to quinone-type inhibitors (menadione and plumbagin) than wild type (WT) cells. In photoautotrophically grown cyanobacterial cells more than 60% of NADPH:quinone-reductase activity, as well as all NADPH:dinoseb-reductase activity, was associated with the function of NQR. NQR activity was observed only in soluble fraction of cyanobacterial cells, but not in membrane fraction. The effects of menadione and menadiol on the reduction of Photosystem I reaction center (P700(+)) after its photooxidation in the presence of DCMU were studied using the EPR spectroscopy. The addition of menadione increased the rate of P700(+) reduction in WT cells, whereas in Ins2 mutant the reduction of P700(+) was strongly inhibited. In the presence of menadiol the reduction of P700(+) was accelerated both in WT and Ins2 mutant cells. These data suggest that NQR protects the cyanobacterial cells from the toxic effect of exogenous quinones by their reduction to hydroquinones. These data may also indicate the probable functional homology of Synechocystis sp. PCC 6803 NQR with mammalian and plant NAD(P)H:quinone oxidoreductases (DT-diaphorases).
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PMID:Role of NAD(P)H:quinone oxidoreductase encoded by drgA gene in reduction of exogenous quinones in cyanobacterium Synechocystis sp. PCC 6803 cells. 1500 Jun 79

Pyocyanin (1-hydroxy-N-methylphenazine) is a cytotoxic pigment secreted by the bacterial species Pseudomonas aeruginosa, which frequently infects the lungs of immunosuppressed patients as well as those with cystic fibrosis. Pyocyanin toxicity results presumably from the ability of the compound to undergo reduction by NAD(P)H and subsequent generation of superoxide and H2O2 directly in the lungs. We report that in the presence of peroxidase mimics, microperoxidase 11, or hemin, pyocyanin undergoes oxidation by H2O2, as evidenced by loss of the pigment's characteristic absorption spectrum and by EPR detection of a free radical metabolite. The oxidation of pyocyanin is irreversible, suggesting an extensive modification of the pigment's phenazine chromophore. Oxidation of pyocyanin was observed also when exogenous H2O2 was replaced by a H2O2-generating system consisting of NADH and the pigment itself. That the oxidation involves the phenolate group of pyocyanin was verified by the observation that a related pigment, phenazine methosulfate, which is devoid of this group, does not undergo oxidation by microperoxidase 11/H2O2. In contrast to intact pyocyanin, oxidized pyocyanin was less efficient in NADH oxidation and stimulation of interleukin-8 release by human alveolar epithelial A549 cells in vitro, suggesting that oxidation of pyocyanin leads to its inactivation. This study demonstrates that pyocyanin may play a dual role in biological systems, first as an oxidant and ROS generator, and second as a substrate for peroxidases, contributing to H2O2 removal. This latter property may cause pyocyanin degradation and inactivation, which may be of considerable biomedical interest.
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PMID:Oxidation of pyocyanin, a cytotoxic product from Pseudomonas aeruginosa, by microperoxidase 11 and hydrogen peroxide. 1513 82

Dihydroorotate dehydrogenase B (DHODB) is a complex iron-sulfur flavoprotein that catalyzes the conversion of dihydroorotate to orotate and the reduction of NAD(+). The enzyme is a dimer of heterodimers containing an FMN, an FAD, and a 2Fe-2S center. UV-visible, EPR, and ENDOR spectroscopies have been used to determine the reduction potentials of the flavins and the 2Fe-2S center and to characterize radicals and their interactions. Reductive titration using dithionite indicates a five-electron capacity for DHODB. The midpoint reduction potential of the 2Fe-2S center (-212 +/- 3 mV) was determined from analysis of absorption data at 540 nm, where absorption contributions from the two flavins are small. The midpoint reduction potentials of the oxidized/semiquinone (E(1)) and semiquinone/hydroquinone (E(2)) couples for the FMN (E(1) = -301 +/- 6 mV; E(2) = -252 +/- 8 mV) and FAD (E(1) = -312 +/- 6 mV; E(2) = -297 +/- 5 mV) were determined from analysis of spectral changes at 630 nm. Corresponding values for the midpoint reduction potentials for FMN (E(1) = -298 +/- 4 mV; E(2) = -259 +/- 5 mV) in the isolated catalytic subunit (subunit D, which lacks the 2Fe-2S center and FAD) are consistent with the values determined for the FMN couples in DHODB. During reductive titration of DHODB, small amounts of the neutral blue semiquinone are observed at approximately 630 nm, consistent with the measured midpoint reduction potentials of the flavins. An ENDOR spectrum of substrate-reduced DHODB identifies hyperfine couplings to proton nuclei similar to those recorded for the blue semiquinone of free flavins in aqueous solution, thus confirming the presence of this species in DHODB. Spectral features observed during EPR spectroscopy of dithionite-reduced DHODB are consistent with the midpoint reduction potentials determined using UV-visible spectroscopy and further identify an unusual EPR signal with very small rhombic anisotropy and g values of 2.02, 1.99, and 1.96. This unusual signal is assigned to the formation of a spin interacting state between the FMN semiquinone species and the reduced 2Fe-2S center. Reduction of DHODB using an excess of NADH or dihydroorotate produces EPR spectra that are distinct from those produced by dithionite. From potentiometric studies, the reduction of the 2Fe-2S center and the reduction of the FMN occur concomitantly. The study provides a detailed thermodynamic framework for electron transfer in this complex iron-sulfur flavoprotein.
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PMID:Thermodynamic basis of electron transfer in dihydroorotate dehydrogenase B from Lactococcus lactis: analysis by potentiometry, EPR spectroscopy, and ENDOR spectroscopy. 1515 83

Infrared spectra of (15)N-enriched preparations of the soluble cytoplasmic NAD(+)-reducing [NiFe]-hydrogenase from Ralstonia eutropha are presented. These spectra, together with chemical analyses, show that the Ni-Fe active site contains four cyanide groups and one carbon monoxide molecule. It is proposed that the active site is a (RS)(2)(CN)Ni(micro-RS)(2)Fe(CN)(3)(CO) centre (R=Cys) and that H(2) activation solely takes place on nickel. One of the two FMN groups (FMN-a) in the enzyme can be reversibly released upon reduction of the enzyme. It is now reported that at longer times also one of the cyanide groups, the one proposed to be bound to the nickel atom, could be removed from the enzyme. This process was irreversible and induced the inhibition of the enzyme activity by oxygen; the enzyme remained insensitive to carbon monoxide. The Ni-Fe active site was EPR undetectable under all conditions tested. It is concluded that the Ni-bound cyanide group is responsible for the oxygen insensitivity of the enzyme.
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PMID:The soluble [NiFe]-hydrogenase from Ralstonia eutropha contains four cyanides in its active site, one of which is responsible for the insensitivity towards oxygen. 1516 70

Thermoanaerobacter tengcongensis is a thermophilic Gram-positive bacterium able to dispose of the reducing equivalents generated during the fermentation of glucose to acetate and CO(2) by reducing H(+) to H(2). A unique combination of hydrogenases, a ferredoxin-dependent [NiFe] hydrogenase and an NADH-dependent Fe-only hydrogenase, were found to be responsible for H(2) formation in this organism. Both enzymes were purified and characterized. The tightly membrane-bound [NiFe] hydrogenase belongs to a small group of complex-I-related [NiFe] hydrogenases and has highest sequence similarity to energy-converting [NiFe] hydrogenase (Ech) from Methanosarcina barkeri. A ferredoxin isolated from Ta. tengcongensis was identified as the physiological substrate of this enzyme. The heterotetrameric Fe-only hydrogenase was isolated from the soluble fraction. It contained FMN and multiple iron-sulfur clusters, and exhibited a typical H-cluster EPR signal after autooxidation. Sequence analysis predicted and kinetic studies confirmed that the enzyme is an NAD(H)-dependent Fe-only hydrogenase. When H(2) was allowed to accumulate in the culture, the fermentation was partially shifted to ethanol production. In cells grown at high hydrogen partial pressure [p(H(2))] the NADH-dependent hydrogenase activity was fourfold lower than in cells grown at low p(H(2)), whereas aldehyde dehydrogenase and alcohol dehydrogenase activities were higher in cells grown at elevated p(H(2)). These results indicate a regulation in response to the p(H(2)).
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PMID:A multisubunit membrane-bound [NiFe] hydrogenase and an NADH-dependent Fe-only hydrogenase in the fermenting bacterium Thermoanaerobacter tengcongensis. 1525 87

Structure and oxidation state of the Ni-Fe cofactor of the NAD-reducing soluble hydrogenase (SH) from Ralstonia eutropha were studied employing X-ray absorption spectroscopy (XAS) at the Ni K-edge, EPR, and FTIR spectroscopy. The SH comprises a nonstandard (CN)Ni-Fe(CN)(3)(CO) site; its hydrogen-cleavage reaction is resistant against inhibition by dioxygen and carbon monoxide. Simulations of the XANES and EXAFS regions of XAS spectra revealed that, in the oxidized SH, the Ni(II) is six-coordinated ((CN)O(3)S(2)); only two of the four conserved cysteines, which bind the Ni in standard Ni-Fe hydrogenases, provide thiol ligands to the Ni. Upon the exceptionally rapid reductive activation of the SH by NADH, an oxygen species is detached from the Ni; hydrogen may subsequently bind to the vacant coordination site. Prolonged reducing conditions cause the two thiols that are remote from the Ni in the native SH to become direct Ni ligands, creating a standardlike Ni(II)(CysS)(4) site, which could be further reduced to form the Ni-C (Ni(III)-H(-)) state. The Ni-C state does not seem to be involved in hydrogen cleavage. Two site-directed mutants (HoxH-I64A, HoxH-L118F) revealed structural changes at their Ni sites and were employed to further dissect the role of the extra CN ligand at the Ni. It is proposed that the predominant coordination by (CN),O ligands stabilizes the Ni(II) oxidation state throughout the catalytic cycle and is a prerequisite for the rapid activation of the SH in the presence of oxygen.
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PMID:Structural and oxidation-state changes at its nonstandard Ni-Fe site during activation of the NAD-reducing hydrogenase from Ralstonia eutropha detected by X-ray absorption, EPR, and FTIR spectroscopy. 1564 82

Toluene 4-monooxygenase (T4MO) is a member of the bacterial multicomponent monooxygenases, an enzyme family that utilizes a soluble diiron hydroxylase to oxidize a variety of hydrocarbons as the initial step in their metabolism. The hydroxylases obtain reducing equivalents from NAD(P)H via an electron transfer chain that is initiated by an oxidoreductase containing an N-terminal ferredoxin domain and C-terminal flavin- and NAD-binding domains. T4moF, the NADH oxidoreductase of T4MO, was expressed as a soluble protein in Escherichia coli BL21(DE3) from the pUC-derived expression vector pRS205. This vector contains a lac promoter instead of a T7 promoter. A three step purification from the soluble cell lysate yielded approximately 1 mg of T4moF per gram of wet cell paste with greater than 90% purity. The purified protein contained 1 mol of FAD and 2 mol of Fe per mol of T4moF; quantitative EPR spectroscopy showed approximately 1 mol of the S=1/2 signal from the reduced [2Fe-2S] cluster per mol of T4moF. Steady state kinetic analysis of p-cresol formation activity treating T4moF as the variable substrate while all other proteins and substrates were held constant gave apparent K(M-) and apparent k(cat)-values of 0.15 microM and 3.0 s(-1), respectively. This expression system and purification allows for the recovery of the soluble oxidoreductase in yields that facilitate further biochemical and structural characterizations.
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PMID:Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase. 1796 5

Three DNA regions carrying genes encoding putative homologs of xanthine dehydrogenases were identified in Escherichia coli, named xdhABC, xdhD, and yagTSRQ. Here, we describe the purification and characterization of gene products of the yagTSRQ operon, a molybdenum-containing iron-sulfur flavoprotein from E. coli, which is located in the periplasm. The 135 kDa enzyme comprised a noncovalent (alpha beta gamma) heterotrimer with a large (78.1 kDa) molybdenum cofactor (Moco)-containing YagR subunit, a medium (33.9 kDa) FAD-containing YagS subunit, and a small (21.0 kDa) 2 x [2Fe2S]-containing YagT subunit. YagQ is not a subunit of the mature enzyme, and the protein is expected to be involved in Moco modification and insertion into YagTSR. Analysis of the form of Moco present in YagTSR revealed the presence of the molybdopterin cytosine dinucleotide cofactor. Two different [2Fe2S] clusters, typical for this class of enzyme, were identified by EPR. YagTSR represents the first example of a molybdopterin cytosine dinucleotide-containing protein in E. coli. Kinetic characterization of the enzyme revealed that YagTSR converts a broad spectrum of aldehydes, with a preference for aromatic aldehydes. Ferredoxin instead of NAD(+) or molecular oxygen was used as terminal electron acceptor. Complete growth inhibition of E. coli cells devoid of genes from the yagTSRQ operon was observed by the addition of cinnamaldehyde to a low-pH medium. This finding shows that YagTSR might have a role in the detoxification of aromatic aldehydes for E. coli under certain growth conditions.
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PMID:A periplasmic aldehyde oxidoreductase represents the first molybdopterin cytosine dinucleotide cofactor containing molybdo-flavoenzyme from Escherichia coli. 1936 56

In prokaryotes, mono-ADP-ribose transfer enzymes represent a family of exotoxins that display activity in a variety of bacterial pathogens responsible for causing disease in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report here that NarE, a putative ADP-ribosylating toxin previously identified from Neisseria meningitidis, which shares structural homologies with Escherichia coli heat labile enterotoxin and toxin from Vibrio cholerae, possesses an iron-sulfur center. The recombinant protein was expressed in E. coli, and when purified at high concentration, NarE is a distinctive golden brown in color. Evidence from UV-visible spectrophotometry and EPR spectroscopy revealed characteristics consistent of an iron-binding protein. The presence of iron was determined by colorimetric method and by an atomic absorption spectrophotometer. To identify the amino acids involved in binding iron, a combination of site-directed mutagenesis and UV-visible and enzymatic assays were performed. All four cysteine residues were individually replaced by serine. Substitution of Cys(67) and Cys(128) into serine caused a drastic reduction in the E(420)/E(280) ratio, suggesting that these two residues are essential for the formation of a stable coordination. This modification led to a consistent loss in ADP-ribosyltransferase activity, while decrease in NAD-glycohydrolase activity was less dramatic in these mutants, indicating that the correct assembly of the iron-binding site is essential for transferase but not hydrolase activity. This is the first observation suggesting that a member of the ADP-ribosyltransferase family contains an Fe-S cluster implicated in catalysis. This observation may unravel novel functions exerted by this class of enzymes.
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PMID:Identification of an iron-sulfur cluster that modulates the enzymatic activity in NarE, a Neisseria meningitidis ADP-ribosyltransferase. 1974 27


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