[FeFe]-Hydrogenase Maturation: HydG-Catalyzed Synthesis of Carbon Monoxide

Shepard, Eric M.; Duffus, Benjamin R.; George, Simon J.; McGlynn, Shawn E.; Challand, Martin R.; Swanson, Kevin D.; Roach, Peter L.; Cramer, Stephen P.; Peters, John W.; Broderick, Joan B.

doi:10.1021/ja1012273

Cited by 140 publications

(206 citation statements)

References 17 publications

(27 reference statements)

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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%

“…HydG-produced CO was detected following a protocol adapted from Shepard et al (11,33). In a 200-μL sealed quartz cuvette were mixed 17 μM MtHydG (or 9 μM Mt-H266N variant) or 9 μM CaHydG (or 20 μM Ca-SxxS variant), 50 mM buffer (Hepes pH 7.4 for CaHydG and Tris pH 8 for MtHydG), 150 mM NaCl, 1 mM sodium dithionite, 0.3 mg/mL bovine hemoglobin (Sigma-Aldrich 51290), and 1 mM tyrosine.…”

Section: Methodsmentioning

confidence: 99%

“…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)(11)(12)(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).…”

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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%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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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 active site of [FeFe]-hydrogenases consists of a 4Fe-4S cluster ligated by four Cys, which is coordinated via a single conserved Cys thiolate sulfur atom to the two Fe core. Each of those two iron ions is attached to a CO and CN − ligand and a bridging CO ligand [134].…”

Section: Ec 1121: Oxidoreductases Of H 2 (Hydrogenases)mentioning

confidence: 99%

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

Vidal

Kelly

Mordaka

et al. 2018

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

235

147

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A B S T R A C T NAD(P)H-dependent oxidoreductases catalyze the reduction or oxidation of a substrate coupled to the oxidation or reduction, respectively, of a nicotinamide adenine dinucleotide cofactor NAD(P)H or NAD(P) + . NAD(P)Hdependent oxidoreductases catalyze a large variety of reactions and play a pivotal role in many central metabolic pathways. Due to the high activity, regiospecificity and stereospecificity with which they catalyze redox reactions, they have been used as key components in a wide range of applications, including substrate utilization, the synthesis of chemicals, biodegradation and detoxification. There is great interest in tailoring NAD(P)H-dependent oxidoreductases to make them more suitable for particular applications. Here, we review the main properties and classes of NAD(P)H-dependent oxidoreductases, the types of reactions they catalyze, some of the main protein engineering techniques used to modify their properties and some interesting examples of their modification and application.

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“…The resultant hydroxybenzyl radical has been characterized in detail using tyrosine isotopologues and freeze quench EPR spectroscopy [12]. Biochemical experiments with HydG identified three tyrosine derived products: the byproduct p-cresol [7], cyanide [13] and carbon monoxide [14]. The last two are incorporated into the [2Fe] H subcluster [15] together with HydG-derived iron atoms [16] The intermediate dehydroglycine is converted to CO and cyanide, a reaction that likely involves a second FeS cluster present in HydG, termed the auxiliary cluster [17].…”

Section: Introductionmentioning

confidence: 99%

Metallocofactor assembly for [FeFe]-hydrogenases

Dinis

Wieckowski

Roach

2016

Current Opinion in Structural Biology

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Hydrogenases are a potential source of environmentally benign bioenergy, using complex cofactors to catalyze the reversible reduction of protons to form hydrogen. AddressesChemistry and the Institute for Life Sciences, University of Southampton, Highfield Campus, Southampton SO17 1BJ, UK.Corresponding author: Peter L. Roach (plr2@soton.ac.uk). AbstractHydrogenases are a potential source of environmentally benign bioenergy, using complex cofactors to catalyze the reversible reduction of protons to form hydrogen. The most active subclass, the [FeFe]-hydrogenases, is dependent on a metallocofactor, the H cluster, that consists of a two iron subcluster ([2Fe] H ) bridging to a classical cubane cluster ([4Fe-4S] H ). The ligands coordinating to the diiron subcluster include an azadithiolate, three carbon monoxides, and two cyanides. To assemble this complex cofactor, three maturase enzymes, HydG, HydE and HydF are required. The biosynthesis of the diatomic ligands proceeds by an unusual fragmentation mechanism, and structural studies in combination with spectroscopic analysis have started to provide insights into the HydG mediated assembly of a [2Fe] H subcluster precursor.

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[FeFe]-Hydrogenase Maturation: HydG-Catalyzed Synthesis of Carbon Monoxide

Cited by 140 publications

References 17 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

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

Metallocofactor assembly for [FeFe]-hydrogenases

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[FeFe]-Hydrogenase Maturation: HydG-Catalyzed Synthesis of Carbon Monoxide

Cited by 140 publications

References 17 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

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

Metallocofactor assembly for [FeFe]-hydrogenases

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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