Mössbauer studies of Escherichia coli biotin synthase: evidence for reversible interconversion between [2Fe‐2S]2+ and [4Fe‐4S]2+ clusters

Bui, Bernadette Tse Sum; Florentin, D.; Marquet, Andrée; Benda, Rüdiger; Trautwein, A. X.

doi:10.1016/s0014-5793(99)01300-9

Cited by 52 publications

(62 citation statements)

References 27 publications

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“…We confirmed this (2Fe-2S) 2+ to (4Fe-4S) 2+ transformation by addition of dithionite (2 equiv) in a glycerol-containing medium using Mössbauer spectroscopy (38). We could observe the liberation of Fe 2+ (≈ 25%) which was not reincorporated into clusters (note that this experiment was done in a glovebox but without oxygen control).…”

Section: Discussionsupporting

confidence: 65%

Fate of the (2Fe-2S)²⁺ Cluster of Escherichia coli Biotin Synthase during Reaction: A Mössbauer Characterization

et al. 2003

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Biotin synthase, the enzyme which catalyzes the last step of the biosynthesis of biotin, contains only (2Fe-2S)(2+) clusters when isolated under aerobic conditions. Previous results showed that reduction by dithionite or photoreduced deazaflavin converts the (2Fe-2S)(2+) to (4Fe-4S)(2+,+). However, until now, no detailed investigation concerning the fate of the (2Fe-2S)(2+) during reduction under assay conditions (NADPH, flavodoxin, flavodoxin reductase) has been realized. Here, we show by Mössbauer spectroscopy on a partially purified fraction overexpressing the enzyme that, in the presence of a S(2)(-) source and Fe(2+), there is conversion of the predominant (2Fe-2S)(2+) clusters into a 1:1 mixture of (2Fe-2S)(2+) and (4Fe-4S)(2+). No change in this cluster composition was observed in the presence of the physiological reducing system. When the reaction was allowed to proceed by addition of the substrate dethiobiotin, the (4Fe-4S)(2+) was untouched whereas the (2Fe-2S)(2+) was degraded into a new species. This is consistent with the hypothesis that the reduced (4Fe-4S) cluster is involved in mediating the cleavage of AdoMet and that the (2Fe-2S)(2+) is the sulfur source for biotin.

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Section: Discussionsupporting

confidence: 65%

Fate of the (2Fe-2S)²⁺ Cluster of Escherichia coli Biotin Synthase during Reaction: A Mössbauer Characterization

et al. 2003

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“…This conclusion is supported by biochemical experiments, electron paramagnetic resonance, resonance Raman and Mössbauer spectroscopies, and the recently solved 3D structure [20]. The (4Fe-4S) centre is highly oxygen sensitive [21,22], and the as-isolated protein contains uniquely the (2Fe-2S) 2+ cluster. We have also found, studying whole cells overexpressing biotin synthase by Mössbauer spectroscopy, that the two clusters were present, although in different proportions [23].…”

Section: The Nature Of the (Fe-s) Clusters In The Active Form Of Biotmentioning

confidence: 70%

Biotin synthase mechanism: an overview

Lotierzo¹,

Bui²,

Florentin³

et al. 2005

Biochemical Society Transactions

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Biotin synthase, a member of the 'radical SAM' (S-adenosylmethionine) family, converts DTB (dethiobiotin) into biotin. The active form of the Escherichia coli enzyme contains two (Fe-S) centres, a (4Fe-4S) and a (2Fe-2S). The (4Fe-4S)2+/+ mediates the electron transfer required for the reductive cleavage of SAM into methionine and a DOA* (deoxyadenosyl radical). Two DOA*, i.e. two SAM molecules, are consumed to activate the positions 6 and 9 of DTB. A direct transfer of isotope from the labelled substrate into DOAH (deoxyadenosine) has been observed with 2H, although not quantitatively, but not with tritium. The source of the sulphur introduced to form biotin is still under debate. We have shown that the (2Fe-2S)2+ cluster can be reconstituted in the apoenzyme with S2- and Fe2+. When S2- was replaced by [34S2-], [35S2-] or Se2-, biotin containing mostly the sulphur isotopes or selenium was obtained. This leads us to favour the hypothesis that the (2Fe-2S) centre is the sulphur donor, which may explain the absence of turnover of the enzyme. DTBSH (9-mercaptodethiobiotin), which already contains the sulphur atom of biotin, was shown to be an alternative substrate of biotin synthase both in vivo and with a crude extract. When this compound was tested with a well-defined in vitro system, the same turnover of one and similar reaction rates were observed for DTB and DTBSH. We postulate that the same intermediate is formed from both substrates.

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“…A first proposal, based on spectroscopic studies (2), envisioned a (2Fe-2S) center which could, under reducing conditions, convert into one (4Fe-4S) center bridging the two subunits of the dimer (4). This proposal was then challenged when we (7) and others (9) demonstrated that anaerobic preparations of BioB could bind as many as four Fe and four S atoms per monomer in the form of (4Fe-4S) 2+/1+ clusters which undergo degradation into (2Fe-2S) clusters upon exposure to air (7)(8)(9)36). Finally, a recent report from Ugulava et al suggested the presence of two different clusters in biotin synthase, a (4Fe-4S) cluster involved in the conversion of dethiobiotin into a radical and a (2Fe-2S) one proposed to be the source of sulfur atoms for incorporation into this radical (10,11).…”

Section: Discussionmentioning

confidence: 99%

Biotin Synthase Is a Pyridoxal Phosphate-Dependent Cysteine Desulfurase

et al. 2002

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Biotin synthase (BioB) is an iron-sulfur dimeric enzyme which catalyzes the last step in biotin synthesis. The reaction consists of the introduction of a sulfur atom into dethiobiotin. It is shown here that BioB displays a significant cysteine desulfurase activity, providing it with the ability to mobilize sulfur from free cysteine. This activity is dependent on pyridoxal 5'-phosphate (PLP) and dithiothreitol and proceeds through a protein-bound persulfide. Like other cysteine desulfurases, BioB binds 1 equiv of PLP. By site-directed mutagenesis, two conserved cysteines, Cys97 and Cys128, are shown to be critical for cysteine desulfuration and are good candidates as the site for a persulfide. Since biotin synthase activity is greatly increased by PLP and cysteine, even though it does not exceed 1 nmol of biotin/nmol of monomer, it is proposed that cysteine desulfuration is intimately linked to biotin synthesis. New scenarios for sulfur insertion into dethiobiotin, in which cysteine persulfides play a key role, are discussed.

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Mössbauer studies of Escherichia coli biotin synthase: evidence for reversible interconversion between [2Fe‐2S]²⁺ and [4Fe‐4S]²⁺ clusters

Abstract: The nature and properties of the iron-sulphur (Fe-S) cluster in as-prepared and reduced biotin synthase of Escherichia coli have been investigated by Mo «ssbauer spectroscopy. Our data clearly demonstrate that in the as-prepared sample, the cluster is present as

Cited by 52 publications

References 27 publications

Fate of the (2Fe-2S)²⁺ Cluster of Escherichia coli Biotin Synthase during Reaction: A Mössbauer Characterization

Fate of the (2Fe-2S)²⁺ Cluster of Escherichia coli Biotin Synthase during Reaction: A Mössbauer Characterization

Biotin synthase mechanism: an overview

Biotin Synthase Is a Pyridoxal Phosphate-Dependent Cysteine Desulfurase

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Mössbauer studies of Escherichia coli biotin synthase: evidence for reversible interconversion between [2Fe‐2S]2+ and [4Fe‐4S]2+ clusters

Abstract: The nature and properties of the iron-sulphur (Fe-S) cluster in as-prepared and reduced biotin synthase of Escherichia coli have been investigated by Mo «ssbauer spectroscopy. Our data clearly demonstrate that in the as-prepared sample, the cluster is present as

Cited by 52 publications

References 27 publications

Fate of the (2Fe-2S)2+ Cluster of Escherichia coli Biotin Synthase during Reaction: A Mössbauer Characterization

Fate of the (2Fe-2S)2+ Cluster of Escherichia coli Biotin Synthase during Reaction: A Mössbauer Characterization

Biotin synthase mechanism: an overview

Biotin Synthase Is a Pyridoxal Phosphate-Dependent Cysteine Desulfurase

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Mössbauer studies of Escherichia coli biotin synthase: evidence for reversible interconversion between [2Fe‐2S]²⁺ and [4Fe‐4S]²⁺ clusters

Fate of the (2Fe-2S)²⁺ Cluster of Escherichia coli Biotin Synthase during Reaction: A Mössbauer Characterization

Fate of the (2Fe-2S)²⁺ Cluster of Escherichia coli Biotin Synthase during Reaction: A Mössbauer Characterization