[31] Determination of FMN and FAD by fluorescence titration with apoflavodoxin

Mayhew, Stephen G.; Wassink, Johannes H.

doi:10.1016/0076-6879(80)66461-1

Cited by 12 publications

(6 citation statements)

References 11 publications

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“…Upon unfolding of PheA2 in 0.5% SDS, the fluorescence intensity of the flavin decreased by almost an order of magnitude. This indicates that the flavin fluorescence quantum yield of PheA2 is comparable to that of free FMN (38) and in the same range as that of lipoamide dehydrogenase, one of the most fluorescent flavoproteins (49). Fig.…”

Section: Resultsmentioning

confidence: 55%

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Phenol Hydroxylase from Bacillus thermoglucosidasius A7, a Two-protein Component Monooxygenase with a Dual Role for FAD

Kirchner

Westphal

Müller

et al. 2003

Journal of Biological Chemistry

125

106

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A novel phenol hydroxylase (PheA) that catalyzes the first step in the degradation of phenol in Bacillus thermoglucosidasius A7 is described. The two-protein system, encoded by the pheA1 and pheA2 genes, consists of an oxygenase (PheA1) and a flavin reductase (PheA2) and is optimally active at 55°C. PheA1 and PheA2 were separately expressed in recombinant Escherichia coli BL21(DE3) pLysS cells and purified to apparent homogeneity. The pheA1 gene codes for a protein of 504 amino acids with a predicted mass of 57.2 kDa. PheA1 exists as a homodimer in solution and has no enzyme activity on its own. PheA1 catalyzes the efficient ortho-hydroxylation of phenol to catechol when supplemented with PheA2 and FAD/NADH. The hydroxylase activity is strictly FAD-dependent, and neither FMN nor riboflavin can replace FAD in this reaction. The pheA2 gene codes for a protein of 161 amino acids with a predicted mass of 17.7 kDa. PheA2 is also a homodimer, with each subunit containing a highly fluorescent FAD prosthetic group. PheA2 catalyzes the NADH-dependent reduction of free flavins according to a Ping Pong Bi Bi mechanism. PheA2 is structurally related to ferric reductase, an NAD(P)H-dependent reductase from the hyperthermophilic Archaea Archaeoglobus fulgidus that catalyzes the flavin-mediated reduction of iron complexes. However, PheA2 displays no ferric reductase activity and is the first member of a newly recognized family of shortchain flavin reductases that use FAD both as a substrate and as a prosthetic group.

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Section: Resultsmentioning

confidence: 55%

“…After removal of the protein precipitate, the nature of the extracted flavin was identified by fluorescence analysis (38) and reverse-phase HPLC (39).…”

Section: Two-component Phenol Hydroxylasementioning

confidence: 99%

Phenol Hydroxylase from Bacillus thermoglucosidasius A7, a Two-protein Component Monooxygenase with a Dual Role for FAD

Kirchner

Westphal

Müller

et al. 2003

Journal of Biological Chemistry

125

106

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“…Again the sample behaved most like FAD, but was still clearly different from it. Treatment of the 0.2 ml sample at room temperature with 50 ,tg of Naja naja phosphodiesterase (Mayhew & Wassink, 1980) confirmed that the unknown flavin was not FAD. The sample showed no change in fluorescence at all on contact for 1 h with the phosphodiesterase, whereas treatment of FAD at a similar concentration for 2 min generated an approx.…”

Section: The Enzyme Prosthetic Groupmentioning

confidence: 88%

Purification and some properties of alcohol oxidase from alkane-grown Candida tropicalis

Dickinson

Wadforth

1992

Biochemical Journal

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Alcohol oxidase was purified to homogeneity from membrane fractions obtained from alkane-grown Candida tropicalis. The enzyme appears to be a dimer of equal-sized subunits of Mr 70000. The purified enzyme is photosensitive and contains flavin-type material which is released by a combination of boiling and proteolytic digestion. The identity of the flavin material is not yet known, but it is not FMN, FAD or riboflavin. The enzyme is most active with dodecan-I-ol, but other long-chain alcohols are also attacked. The enzyme shows a weak, but significant activity towards long-chain aldehydes. Detailed kinetic studies with decan-1-ol as substrate suggest a group-transfer (Ping-Pong)-type mechanism of catalysis.

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“…Simulation of EPR spectra was achieved as in [18]. The flavin moiety in HCR was analysed after quantitative extraction of the cofactor by unfolding and precipitation of the protein with 5% (w/v) trichloroacetic acid [19]. For identification the isolated cofactor was incubated with snake‐venom phosphodiesterase ( Naja naja venom); during this incubation, fluorescence emission spectra between 480 and 650 nm (λ exc = 450 nm) were recorded.…”

Section: Methodsmentioning

confidence: 99%

The hybrid‐cluster protein (‘prismane protein’) from Escherichia coli

Berg

Hagen

Dongen

2000

European Journal of Biochemistry

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Hybrid-cluster proteins (`prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe±4S] cubane cluster with spin-admixed S = 3/2 ground-state paramagnetism and a novel type of hybrid [4Fe±2S±2O] cluster, which can attain four redox states.Genomic sequencing reveals that genes encoding putative hybrid-cluster proteins are present in a range of bacterial and archaeal species. In this paper we describe the isolation and spectroscopic characterization of the hybrid-cluster protein from Escherichia coli. EPR spectroscopy shows the presence of a hybrid cluster in the E. coli protein with characteristics similar to those in the proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid-cluster protein, however, give evidence for the presence of a [2Fe±2S] cluster instead of a [4Fe±4S] cluster. The hcp gene encoding the hybrid-cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and archaea, in a small operon with a gene encoding a putative NADH oxidoreductase. This NADH oxidoreductase was also isolated and shown to contain FAD and a [2Fe±2S] cluster as cofactors. It catalysed the reduction of the hybrid-cluster protein with NADH as an electron donor. Midpoint potentials (25 8C, pH 7.5) for the Fe/S clusters in both proteins indicate that electrons derived from the oxidation of NADH (E m NADH/NAD + couple: 2320 mV) are transferred along the [2Fe±2S] cluster of the NADH oxidoreductase (E m = ±220 mV) and the [2Fe±2S] cluster of the hybrid-cluster protein (E m = ±35 mV) to the hybrid cluster (E m = ±50, +85 and +365 mV for the three redox transitions).The physiological function of the hybrid-cluster protein has not yet been elucidated. The protein is only detected in the facultative anaerobes E. coli and Morganella morganii after cultivation under anaerobic conditions in the presence of nitrate or nitrite, suggesting a role in nitrate-and/or nitrite respiration.Keywords: EPR; hybrid-cluster protein (`prismane protein'); NAD(H) oxidoreductase; nitrate regulation; redox titration.The`hybrid-cluster protein' (HCP, formerly named`prismane protein') was initially purified from species of the strictly anaerobic sulfate-reducing bacterial genus Desulfovibrio: D. vulgaris (Hildenborough) [1,2] and D. desulfuricans ATCC 27774 [3,4]. This soluble cytoplasmic protein has been extensively studied because of the unusual properties of its redox-active iron clusters. Initial characterization with EPR and Mo Èssbauer spectroscopy suggested the presence of an Fe/S cluster with magnetic properties similar to those of synthetic [6Fe±6S] model compounds (`prismane' clusters; hence the name`prismane protein' was initially proposed for this protein) [1,5].Recently, the three-dimensional structure of the D. vulgaris HCP' at 1.7 A Ê resolution was obtained by X-ray crystallography [6]. The structure reveal...

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[31] Determination of FMN and FAD by fluorescence titration with apoflavodoxin

Cited by 12 publications

References 11 publications

Phenol Hydroxylase from Bacillus thermoglucosidasius A7, a Two-protein Component Monooxygenase with a Dual Role for FAD

Phenol Hydroxylase from Bacillus thermoglucosidasius A7, a Two-protein Component Monooxygenase with a Dual Role for FAD

Purification and some properties of alcohol oxidase from alkane-grown Candida tropicalis

The hybrid‐cluster protein (‘prismane protein’) from Escherichia coli

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