Further Characterization of the [FeFe]‐Hydrogenase Maturase HydG

Tron, Cécile; Cherrier, M.V.; Amara, Patricia; Martin, Lydie; Fauth, François; Fraga, Edmundo; Correard, Mélanie; Fontecave, Marc; Nicolet, Yvain; Fontecilla‐Camps, Juan C.

doi:10.1002/ejic.201001101

Cited by 25 publications

(39 citation statements)

References 37 publications

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“…S2), HydG variants lacking a functional second cluster are expected to produce − :CO 2 H-derived formate instead of CO and water. Points (i)-(iii) have already been addressed (11)(12)(13)26) and (ii) and (iii) are further confirmed by our results presented below. Conversely, production of the − :CO 2 H intermediate, measured indirectly as HCO 2 − (formate), has been investigated here for the first time to our knowledge.…”

Section: Significancesupporting

confidence: 87%

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CO and CN ⁻ syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events

Pagnier

Martin

Zeppieri

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The synthesis and assembly of the active site [FeFe] unit of [FeFe]-hydrogenases require at least three maturases. The radical S-adenosyl-L-methionine HydG, the best characterized of these proteins, is responsible for the synthesis of the hydrogenase CO and CN − ligands from tyrosine-derived dehydroglycine (DHG). We speculated that CN − and the CO precursor − :CO 2 H may be generated through an elimination reaction. We tested this hypothesis with both wild type and HydG variants defective in second ironsulfur cluster coordination by measuring the in vitro production of CO, CN − , and − :CO 2 H-derived formate. We indeed observed formate production under these conditions. We conclude that HydG is a multifunctional enzyme that produces DHG, CN − , and CO at three well-differentiated catalytic sites. We also speculate that homocysteine, cysteine, or a related ligand could be involved in Fe(CO) x (CN) y transfer to the HydF carrier/scaffold. (4) showed that the active site is composed of a conventional [4Fe-4S] cubane connected by a cysteine thiolate to a binuclear FeFe unit, in which each iron ion is terminally coordinated by one CN − ligand and one CO ligand and by a third CO molecule that bridges the two metals (5). Unexpectedly, we also found that a small molecule first postulated (6), and now indirectly confirmed (7), to be dithiomethylamine (DTMA) bridges the two Fe ions (Fig. 1).The [4Fe-4S] cubane bridged to the binuclear [FeFe] unit has been collectively called the H-cluster (1). Work from several laboratories has shown that the maturation of the [FeFe] center requires at least three protein maturases: HydF that has GTPase activity and appears to be both a [FeFe] center scaffold and carrier (8, 9), HydG that synthesizes CO and CN − from tyrosine (10-13), and HydE that, by elimination, should be involved in the synthesis of the DTMA bridge (14, 15). Both HydE and HydG are members of the large radical S-adenosyl-L-methionine (SAM) protein family (16,17). With the recent reports of HydG crystal structures from Carboxydothermus hydrogenoformans (Ch) by us (18) and from Thermoanaerobacter italicus (Ti) by Dinis et al. (19), X-ray models are now available for the three maturases (20, 21); however, unambiguous structure-function relationships have been proposed only in the case of HydG. Indeed, site-directed mutational studies have shown that CO and CN − syntheses are affected by either the deletion of the maturase C-terminal region, where a second iron-sulfur cluster binds (22), or Cys-to-Ser mutations in its corresponding CxxCx 22 C binding motif (10, 13). In addition, it has been shown that HydG synthesizes Fe(CO) x (CN) y precursors (x = 1 or 2; y = 1) of the [FeFe] catalytic unit (23). The two HydG crystal structures are very similar at the SAM and [4Fe-4S] cluster-containing (β/α) 8 TIM-like barrel, common to several radical SAM proteins (16) (Fig. 2). Conversely, there are significant differences in the composition of the extra C-terminal second (s) iron-sulfur cluster. In our crystals, ChHydG lacks th...

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Section: Significancesupporting

confidence: 87%

“…Δ CTD = mutant with a C-terminal domain deletion (III). Its tyrosine binding affinity at I is reduced (26). + and -signs signify increased and diminished production, respectively (or consumption in the case of tyrosine).…”

Section: Significancementioning

confidence: 99%

CO and CN ⁻ syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events

Pagnier

Martin

Zeppieri

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Conversely, these mutations completely abolish CO synthesis, indicating that this process requires the second [Fe 4 S 4 ] cluster (Fig. 2, A and C) (27,28). We concluded that, although CO and CN Ϫ are produced at similar rates (12,13), their syntheses are not simultaneous and are likely to occur at different locations of the protein (27,28).…”

Section: Activity Of [Fefe]-hydrogenase Maturasesmentioning

confidence: 81%

“…These studies show that the C-terminal stretch that coordinates the second [Fe 4 S 4 ] cluster forms a domain that interacts extensively with the bottom of the TIM barrel (Fig. 2B) (28). The truncation mutant is still capable of cleaving tyrosine to produce p-cresol (28).…”

Section: Activity Of [Fefe]-hydrogenase Maturasesmentioning

confidence: 95%

“…2, A and C) (27,28). We concluded that, although CO and CN Ϫ are produced at similar rates (12,13), their syntheses are not simultaneous and are likely to occur at different locations of the protein (27,28). A comparison of HydG and ThiH, using the HydE and BioB structures for amino acid sequence threading, indicates that the residues lining the TIM barrel cavity can be grouped into three layers (28): one layer common to all radical SAM enzymes involved in [Fe 4 S 4 ] binding and SAM cleavage, a second layer common to HydG and ThiH and probably involved in tyrosine binding, and a third layer containing residues that are different in ThiH and HydG (Fig.…”

Section: Activity Of [Fefe]-hydrogenase Maturasesmentioning

confidence: 99%

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Structure-Function Relationships in [FeFe]-Hydrogenase Active Site Maturation

Nicolet

Fontecilla‐Camps

2012

Journal of Biological Chemistry

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Since the discovery that, despite the active site complexity, only three gene products suffice to obtain active recombinant [FeFe]-hydrogenase, significant light has been shed on this process. Both the source of the CO and CN ؊ ligands to iron and the assembly site of the catalytic subcluster are known, and an apo structure of HydF has been published recently. However, the nature of the substrate(s) for the synthesis of the bridging dithiolate ligand to the subcluster remains to be established.From both spectroscopy and model chemistry, it is predicted that an amine function in this ligand plays a central role in catalysis, acting as a base in the heterolytic cleavage of hydrogen.

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[ FeFe ]‐Hydrogenase Cofactor Assembly

Shepard

Byer

Boyd

et al. 2004

Encyclopedia of Inorganic and Bioinorganic Chemistry

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This article highlights recent advances in our understanding of the biosynthetic pathway for active site H‐cluster assembly in [ FeFe ]‐hydrogenases. The H‐cluster is composed of a [ 4Fe–4S ] cubane bridged via one cysteine thiolate to a 2Fe subcluster; the 2Fe subcluster is further coordinated by carbon monoxide, cyanide, and bridging dithiolate ligands. Biosynthesis of the H‐cluster occurs through stepwise modification of simple Fe–S cluster precursors; these chemical modifications are carried out by three gene products denoted HydE , HydF , and HydG . Maturation of the [ FeFe ]‐hydrogenase requires the presence of a preformed [ 4Fe–4S ] cluster, indicating that HydE , HydF , and HydG are directed toward the biosynthesis of the 2Fe subcluster. HydE and HydG are both radical S ‐adenosylmethionine ( SAM ) enzymes and utilize a CX 3 CX 2 C motif to bind site‐differentiated [ 4Fe–4S ] 2+/+ clusters that promote the reductive cleavage of SAM into methionine and a 5′‐deoxyadenosyl radical responsible for hydrogen atom abstraction from substrate. In an extraordinary biochemical reaction, HydG uses this chemistry to catalyze the radical degradation of tyrosine into p ‐cresol and a glycine‐like intermediate; an accessory, C‐terminal [ 4Fe–4S ] cluster then degrades the latter intermediate into cyanide and carbon monoxide. HydE is presumed to be responsible for bridging dithiolate ligand biosynthesis from an unknown substrate. HydF contains an N‐terminal GTPase domain and a C‐terminal Fe–S ‐cluster‐binding domain and binds both [ 2Fe–2S ] and [ 4Fe–4S ] clusters; evidence suggests that the former cluster is modified by HydE and HydG to form a 2Fe H‐cluster precursor on HydF . The role of GTP hydrolysis in H‐cluster maturation remains largely unexplained; however, it may be related to gating the protein–protein interactions between HydF and HydE and HydG . The reactions catalyzed by HydE and HydG can be thought of as specific ligand modification events designed to fine‐tune H‐cluster reactivity; not only do the chemical transformations themselves have direct links to reactions important on early Earth, but radical SAM enzymes with their canonical CX 3 CX 2 C motif are likely very ancient. The modern diversity that is observed in chemical reactions catalyzed by the radical SAM superfamily can be explained evolutionarily through the recruitment of distinct protein domains that impart specific substrate activation capabilities. Recruitment of these domains is discussed in the context of HydE and HydG and important links are made to the nitrogenase system where intriguing parallels between [ FeFe ]‐hydA H‐cluster biosynthesis and nitrogenase FeMo ‐cofactor assembly clearly exist.

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Further Characterization of the [FeFe]‐Hydrogenase Maturase HydG

Cited by 25 publications

References 37 publications

CO and CN ⁻ syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events

CO and CN ⁻ syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events

Structure-Function Relationships in [FeFe]-Hydrogenase Active Site Maturation

[ FeFe ]‐Hydrogenase Cofactor Assembly

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Further Characterization of the [FeFe]‐Hydrogenase Maturase HydG

Cited by 25 publications

References 37 publications

CO and CN − syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events

CO and CN − syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events

Structure-Function Relationships in [FeFe]-Hydrogenase Active Site Maturation

[ FeFe ]‐Hydrogenase Cofactor Assembly

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Product

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CO and CN ⁻ syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events

CO and CN ⁻ syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events