Isolation and characterization of the pyruvate-ferredoxin oxidoreductase from the sulfate-reducing bacterium Desulfovibrio africanus

Pieulle, Lætitia; Guigliarelli, Bruno; Asso, Marcel; Dole, François; Bernadac, Alain; Hatchikian, E.C.

doi:10.1016/0167-4838(95)00029-t

Cited by 105 publications

(157 citation statements)

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“…Thus, the electron from HE-TPP must have been transferred from cluster A to cluster B. The pyruvate/acetyl-CoA half-cell reaction is at -520 mV, and the [4Fe-4S] centers in the D. africanus PFOR exhibit midpoint redox potentials of -540, -515, and -390 mV, 14 although these potentials have not been assigned to specific clusters.…”

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confidence: 99%

Pulsed Electron Paramagnetic Resonance Experiments Identify the Paramagnetic Intermediates in the Pyruvate Ferredoxin Oxidoreductase Catalytic Cycle

et al. 2006

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Pyruvate ferredoxin oxidoreductase (PFOR) 1 is a thiamine pyrophosphate (TPP)-dependent enzyme that catalyzes the anaerobic oxidation of pyruvate (with CoA) to acetyl-CoA, CO 2 , and two electrons that are transferred to ferredoxin (eq 1). 2 In this paper, the PFOR electrontransfer mechanism is examined. Early steps in the catalytic cycle follow the Breslow 3 mechanism of TPP activation. After deprotonation, the carbanion at C-2 of TPP reacts with pyruvate to form 2-lactyl-TPP, which decarboxylates to generate CO 2 and the resonant enamine/carbanion intermediate, hydroxyethylidene-TPP (HE-TPP). The fate of this intermediate differs among the TPP-dependent enzymes. In PFOR, oxidation of the intermediate is linked to reduction of an external electron acceptor, for example, the clostridial ferredoxin, which contains two [4Fe-4S] clusters.(1)After formation of HE-TPP, the next step in the PFOR mechanism is one-electron transfer from HE-TPP to one of three [4Fe-4S] 2+ clusters, which generates a paramagnetic [4Fe-4S] 1+ cluster and an HE-TPP radical. 4 This paramagnetic intermediate forms at a rate constant of 140 s −1 at 10 °C (~3000 s −1 at the growth temperature of 55 °C). 4 On the basis of the PFOR structure, 5 cluster A, located only 9.5 Å from the thiazole sulfur of HE-TPP, is one of three [4Fe-4S] clusters that are separated by 10-12 Å (Figure 1). 6 Since biological electron transfer is facile whenever the donor and acceptor are within ~15 Å, clusters A (proximal), B (medial), and C (distal, ~4 Å from the surface) are ideally positioned to sequentially transfer electrons to the surface and the external ferredoxin. Cluster A is logically the first to be reduced, since clusters B and C are >20 Å from the radical center on HE-TPP. In the pulsed ELDOR experiment, the primary electron spin-echo (ESE) signal of the [4Fe-4S] 1+ cluster was observed, while the pumping microwave pulse was in resonance with the HE-TPP radical. The Fourier transform (FT) spectrum of the time-domain ELDOR trace exhibits a prominent peak at 1.5 MHz and a smaller feature at 2.7 MHz (trace 1 in Figure 2). Although this spectral shape is reasonably close to that expected in a situation of complete orientational disorder, some orientational selectivity caused by the g-anisotropy of the [4Fe-4S] 1+ center cannot be excluded. To remove the ambiguity, a RIDME experiment was performed. 11 In this experiment, we observed a refocused stimulated ESE signal of the HE-TPP radical, while the longitudinal relaxation of the [4Fe-4S] 1+ center (instead of pumping at a different microwave frequency) served the purpose of modifying the local magnetic field for the radical spin. Since the [4Fe-4S] 1+ center at any orientation is subject to longitudinal relaxation, 12 there is no orientational selectivity in the RIDME experiment.The RIDME experiment was performed at 13 and 4.2 K. At 13 K, the cluster relaxes much more rapidly than at 4.2 K. The difference between the two stimulated ESE spectra gave the RIDME spectrum shown by trace 2 in Figure 2. The only reli...

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Pulsed Electron Paramagnetic Resonance Experiments Identify the Paramagnetic Intermediates in the Pyruvate Ferredoxin Oxidoreductase Catalytic Cycle

et al. 2006

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“…In contrast, Desulfovibrio africanus PFO has been shown to contain three [4Fe-4S] centers per subunit [4].…”

Section: Discussionmentioning

confidence: 94%

“…The spectrum of Fig. 1A also shows a very weak isotropic signal, most likely arising from a radical, but without the hyperfine structure contributed by a-ketoacids [4]. All spectra of Fig.…”

Section: Molecular Propertiesmentioning

confidence: 89%

“…The quaternary structure and partial sequence of C. pasteurianum PFO resemble those of similar enzymes from bacteria or unicellular eucarya [4,[13][14][15][16], but differ from the heterodimers or heterotetramers found in archaea [3,10,17,18].…”

Section: Discussionmentioning

confidence: 99%

“…The enzyme responsible for the oxidation of pyruvate in several bacteria, archaea or protists is pyruvate-ferredoxin oxidoreductase (PFO). PFO contains thiamine pyrophosphate (TPP) and iron-sulfur centers ( [3,4] and references therein), but variations in quaternary structure, cofactor(s) content or sensitivity to inhibitors have been observed depending on the source of the enzyme. This report presents the main molecular properties of the enzyme purified from Clostridium pasteurianum, which reveal mechanistic differences among the enzymes from various sources.…”

Section: Introductionmentioning

confidence: 99%

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Molecular mechanism of pyruvate‐ferredoxin oxidoreductases based on data obtained with the Clostridium pasteurianum enzyme

et al. 1996

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Pyruvate–Ferredoxin Oxidoreductase

Chabrière¹,

Cavazza²,

Contreras‐Martel³

2004

Handbook of Metalloproteins

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Introduction. Giardia intestinalis is a unicellular parasite of worldwide distribution. It causes an intestinal illness known as giardiasis, and it is probably the earliest diverging eukaryotic microorganism. Previously, changes have been reported in the expression of mRNAs at several stages of the life cycle; however specific enzymatic activity changes have not been explored. Objective. The expression of pyruvate ferredoxin oxidoreductase (PFOR) and alcohol dehydrogenase E (ADHE) enzymes was measured in cyst and trophozoite stages, and during the excystation process. Materials and methods. Recombinant proteins were generated for PFOR and ADHE to be used as antigens in the production of polyclonal antibodies for the detection of native proteins by Western Blot. The enzymatic activity of ADHE and glutamate dehydrogenase (GDH) was evaluated by spectrophotometric assays. Results. PFOR (139 kDa) and ADHE (97 kDa) proteins were detected in trophozoites, but not in cysts. During excystation, ADHE protein was detected after the first phase of induction, but the PFOR protein appeared only after the second phase. This indicated that both proteins were synthesized during excystation, although at different times. ADHE enzymatic activity was present only in trophozoites and not in cysts whereas GDH activity was detected in both stages. Conclusion. These results conclusively showed that PFOR and ADHE enzymes were translated during the excystation process and is strong evidence that active protein synthesis was occurring during excystation.

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Isolation and characterization of the pyruvate-ferredoxin oxidoreductase from the sulfate-reducing bacterium Desulfovibrio africanus

Cited by 105 publications

References 51 publications

Pulsed Electron Paramagnetic Resonance Experiments Identify the Paramagnetic Intermediates in the Pyruvate Ferredoxin Oxidoreductase Catalytic Cycle

Pulsed Electron Paramagnetic Resonance Experiments Identify the Paramagnetic Intermediates in the Pyruvate Ferredoxin Oxidoreductase Catalytic Cycle

Molecular mechanism of pyruvate‐ferredoxin oxidoreductases based on data obtained with the Clostridium pasteurianum enzyme

Pyruvate–Ferredoxin Oxidoreductase

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