Role of Ammonia Lyases in the Synthesis of the Dithiomethylamine Ligand During [FeFe]-hydrogenase Maturation.

The generation of an active [FeFe]-hydrogenase requires the synthesis of a complex metal center, the H-cluster, by three dedicated maturases: the radical S-adenosyl-l-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. A key step of [FeFe]-hydrogenase maturation is the synthesis of the dithiomethylamine (DTMA) bridging ligand, a process recently shown to involve the aminomethyl-lipoyl-H-protein from the glycine cleavage system, whose methylamine group originates from serine and ammonium. Here we use functional assays together with electron paramagnetic resonance and electron-nuclear double resonance spectroscopies to show that serine or aspartate together with their respective ammonia-lyase enzymes can provide the nitrogen for DTMA biosynthesis during in vitro [FeFe]-hydrogenase maturation. We also report bioinformatic analysis of the hyd operon, revealing a strong association with genes encoding ammonia-lyases, suggesting important biochemical and metabolic connections. Together, our results provide evidence that ammonia-lyases play an important role in [FeFe]-hydrogenase maturation by delivering the ammonium required for dithiomethylamine ligand synthesis.

Semisynthetic maturation of [FeFe]-hydrogenase using [Fe2(µ-SH)2(CN)2(CO)4]2-: key roles for HydF and GTP.

Here we describe maturation of the [FeFe]-hydrogenase from its [4Fe-4S]-bound precursor state by using the synthetic complex [Fe2(µ-SH)2(CN)2(CO)4]2- together with HydF and components of the glycine cleavage system, but in the absence of the maturases HydE and HydG. This semisynthetic and fully-defined maturation provides new insights into the nature of H-cluster biosynthesis.

[FeFe]-Hydrogenase In Vitro Maturation.

The [FeFe]-hydrogenase H-cluster is a complex organometallic cofactor whose assembly and installation requires three dedicated accessory proteins referred to as HydE, HydF, and HydG. The roles of these maturases and the precise mechanisms by which they synthesize and insert the H-cluster are not fully understood. This Minireview will focus on new insights into the [FeFe]-hydrogenase maturation process that have been provided by in vitro approaches in which the biosynthetic pathway has been partially or fully reconstructed using semisynthetic and enzyme-based approaches. Specifically, the application of these in vitro, semisynthetic, and fully defined approaches has shed light on the roles of individual maturation enzymes, the nature of H-cluster assembly intermediates, the molecular precursors of H-cluster ligands, and the sequence of steps involved in [FeFe]-hydrogenase maturation.

Mechanism of Radical S-Adenosyl-l-methionine Adenosylation: Radical Intermediates and the Catalytic Competence of the 5'-Deoxyadenosyl Radical.

Radical S-adenosyl-l-methionine (SAM) enzymes employ a [4Fe-4S] cluster and SAM to initiate diverse radical reactions via either H-atom abstraction or substrate adenosylation. Here we use freeze-quench techniques together with electron paramagnetic resonance (EPR) spectroscopy to provide snapshots of the reaction pathway in an adenosylation reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme on a peptide substrate containing a dehydroalanine residue in place of the target glycine. The reaction proceeds via the initial formation of the organometallic intermediate Ω, as evidenced by the characteristic EPR signal with g∥ = 2.035 and g⊥ = 2.004 observed when the reaction is freeze-quenched at 500 ms. Thermal annealing of frozen Ω converts it into a second paramagnetic species centered at giso = 2.004; this second species was generated directly using freeze-quench at intermediate times (∼8 s) and unequivocally identified via isotopic labeling and EPR spectroscopy as the tertiary peptide radical resulting from adenosylation of the peptide substrate. An additional paramagnetic species observed in samples quenched at intermediate times was revealed through thermal annealing while frozen and spectral subtraction as the SAM-derived 5'-deoxyadenosyl radical (5'-dAdo•). The time course of the 5'-dAdo• and tertiary peptide radical EPR signals reveals that the former generates the latter. These results thus support a mechanism in which Ω liberates 5'-dAdo• by Fe-C5' bond homolysis, and the 5'-dAdo• attacks the dehydroalanine residue of the peptide substrate to form the adenosylated peptide radical species. The results thus provide a picture of a catalytically competent 5'-dAdo• intermediate trapped just prior to reaction with the substrate.

[FeFe]-Hydrogenase: Defined Lysate-Free Maturation Reveals a Key Role for Lipoyl-H-Protein in DTMA Ligand Biosynthesis.

Maturation of [FeFe]-hydrogenase (HydA) involves synthesis of a CO, CN- , and dithiomethylamine (DTMA)-coordinated 2Fe subcluster that is inserted into HydA to make the active hydrogenase. This process requires three maturation enzymes: the radical S-adenosyl-l-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. In vitro maturation with purified maturation enzymes has been possible only when clarified cell lysate was added, with the lysate presumably providing essential components for DTMA synthesis and delivery. Here we report maturation of [FeFe]-hydrogenase using a fully defined system that includes components of the glycine cleavage system (GCS), but no cell lysate. Our results reveal for the first time an essential role for the aminomethyl-lipoyl-H-protein of the GCS in hydrogenase maturation and the synthesis of the DTMA ligand of the H-cluster. In addition, we show that ammonia is the source of the bridgehead nitrogen of DTMA.

HydG, the "dangler" iron, and catalytic production of free CO and CN-: implications for [FeFe]-hydrogenase maturation.

The organometallic H-cluster of the [FeFe]-hydrogenase consists of a [4Fe-4S] cubane bridged via a cysteinyl thiolate to a 2Fe subcluster ([2Fe]H) containing CO, CN-, and dithiomethylamine (DTMA) ligands. The H-cluster is synthesized by three dedicated maturation proteins: the radical SAM enzymes HydE and HydG synthesize the non-protein ligands, while the GTPase HydF serves as a scaffold for assembly of [2Fe]H prior to its delivery to the [FeFe]-hydrogenase containing the [4Fe-4S] cubane. HydG uses l-tyrosine as a substrate, cleaving it to produce p-cresol as well as the CO and CN- ligands to the H-cluster, although there is some question as to whether these are formed as free diatomics or as part of a [Fe(CO)2(CN)] synthon. Here we show that Clostridium acetobutylicum (C.a.) HydG catalyzes formation of multiple equivalents of free CO at rates comparable to those for CN- formation. Free CN- is also formed in excess molar equivalents over protein. A g = 8.9 EPR signal is observed for C.a. HydG reconstituted to load the 5th "dangler" iron of the auxiliary [4Fe-4S][FeCys] cluster and is assigned to this "dangler-loaded" cluster state. Free CO and CN- formation and the degree of activation of [FeFe]-hydrogenase all occur regardless of dangler loading, but are increased 10-35% in the dangler-loaded HydG; this indicates the dangler iron is not essential to this process but may affect relevant catalysis. During HydG turnover in the presence of myoglobin, the g = 8.9 signal remains unchanged, indicating that a [Fe(CO)2(CN)(Cys)] synthon is not formed at the dangler iron. Mutation of the only protein ligand to the dangler iron, H272, to alanine nearly completely abolishes both free CO formation and hydrogenase activation, however results show this is not due solely to the loss of the dangler iron. In experiments with wild type and H272A HydG, and with different degrees of dangler loading, we observe a consistent correlation between free CO/CN- formation and hydrogenase activation. Taken in full, our results point to free CO/CN-, but not an [Fe(CO)2(CN)(Cys)] synthon, as essential species in hydrogenase maturation.

S-Adenosyl-l-ethionine is a Catalytically Competent Analog of S-Adenosyl-l-methione (SAM) in the Radical SAM Enzyme HydG.

Radical S-adenosyl-l-methionine (SAM) enzymes initiate biological radical reactions with the 5'-deoxyadenosyl radical (5'-dAdo•). A [4Fe-4S]+ cluster reductively cleaves SAM to form the Ω organometallic intermediate in which the 5'-deoxyadenosyl moiety is directly bound to the unique iron of the [4Fe-4S] cluster, with subsequent liberation of 5'-dAdo•. Here we present synthesis of the SAM analog S-adenosyl-l-ethionine (SAE) and show SAE is a mechanistically-equivalent SAM-alternative for HydG, both supporting enzymatic turnover of substrate tyrosine and forming the organometallic intermediate Ω. Photolysis of SAE bound to HydG forms an ethyl radical trapped in the active site. The ethyl radical withstands prolonged storage at 77 K and its EPR signal is only partially lost upon annealing at 100 K, making it significantly less reactive than the methyl radical formed by SAM photolysis. Upon annealing above 77K, the ethyl radical adds to the [4Fe-4S]2+ cluster, generating an ethyl-[4Fe-4S]3+ organometallic species termed ΩE.

Active-Site Controlled, Jahn-Teller Enabled Regioselectivity in Reductive S-C Bond Cleavage of S-Adenosylmethionine in Radical SAM Enzymes.

Catalysis by canonical radical S-adenosyl-l-methionine (SAM) enzymes involves electron transfer (ET) from [4Fe-4S]+ to SAM, generating an R3S0 radical that undergoes regioselective homolytic reductive cleavage of the S-C5' bond to generate the 5'-dAdo· radical. However, cryogenic photoinduced S-C bond cleavage has regioselectively yielded either 5'-dAdo· or ·CH3, and indeed, each of the three SAM S-C bonds can be regioselectively cleaved in an RS enzyme. This diversity highlights a longstanding central question: what controls regioselective homolytic S-C bond cleavage upon SAM reduction? We here provide an unexpected answer, founded on our observation that photoinduced S-C bond cleavage in multiple canonical RS enzymes reveals two enzyme classes: in one, photolysis forms 5'-dAdo·, and in another it forms ·CH3. The identity of the cleaved S-C bond correlates with SAM ribose conformation but not with positioning and orientation of the sulfonium center relative to the [4Fe-4S] cluster. We have recognized the reduced-SAM R3S0 radical is a (2E) state with its antibonding unpaired electron in an orbital doublet, which renders R3S0 Jahn-Teller (JT)-active and therefore subject to vibronically induced distortion. Active-site forces induce a JT distortion that localizes the odd electron in a single priority S-C antibond, which undergoes regioselective cleavage. In photolytic cleavage those forces act through control of the ribose conformation and are transmitted to the sulfur via the S-C5' bond, but during catalysis thermally induced conformational changes that enable ET from a cluster iron generate dominant additional forces that specifically select S-C5' for cleavage. This motion also can explain how 5'-dAdo· subsequently forms the organometallic intermediate Ω.

Radical SAM Enzyme Spore Photoproduct Lyase: Properties of the Ω Organometallic Intermediate and Identification of Stable Protein Radicals Formed during Substrate-Free Turnover.

Spore photoproduct lyase is a radical S-adenosyl-l-methionine (SAM) enzyme with the unusual property that addition of SAM to the [4Fe-4S]1+ enzyme absent substrate results in rapid electron transfer to SAM with accompanying homolytic S-C5' bond cleavage. Herein, we demonstrate that this unusual reaction forms the organometallic intermediate Ω in which the unique Fe atom of the [4Fe-4S] cluster is bound to C5' of the 5'-deoxyadenosyl radical (5'-dAdo•). During catalysis, homolytic cleavage of the Fe-C5' bond liberates 5'-dAdo• for reaction with substrate, but here, we use Ω formation without substrate to determine the thermal stability of Ω. The reaction of Geobacillus thermodenitrificans SPL (GtSPL) with SAM forms Ω within ∼15 ms after mixing. By monitoring the decay of Ω through rapid freeze-quench trapping at progressively longer times we find an ambient temperature decay time of the Ω Fe-C5' bond of τ ≈ 5-6 s, likely shortened by enzymatic activation as is the case with the Co-C5' bond of B12. We have further used hand quenching at times up to 10 min, and thus with multiple SAM turnovers, to probe the fate of the 5'-dAdo• radical liberated by Ω. In the absence of substrate, Ω undergoes low-probability conversion to a stable protein radical. The WT enzyme with valine at residue 172 accumulates a Val•; mutation of Val172 to isoleucine or cysteine results in accumulation of an Ile• or Cys• radical, respectively. The structures of the radical in WT, V172I, and V172C variants have been established by detailed EPR/DFT analyses.

Paradigm Shift for Radical S-Adenosyl-l-methionine Reactions: The Organometallic Intermediate Ω Is Central to Catalysis.

Radical S-adenosyl-l-methionine (SAM) enzymes comprise a vast superfamily catalyzing diverse reactions essential to all life through homolytic SAM cleavage to liberate the highly reactive 5'-deoxyadenosyl radical (5'-dAdo·). Our recent observation of a catalytically competent organometallic intermediate Ω that forms during reaction of the radical SAM (RS) enzyme pyruvate formate-lyase activating-enzyme (PFL-AE) was therefore quite surprising, and led to the question of its broad relevance in the superfamily. We now show that Ω in PFL-AE forms as an intermediate under a variety of mixing order conditions, suggesting it is central to catalysis in this enzyme. We further demonstrate that Ω forms in a suite of RS enzymes chosen to span the totality of superfamily reaction types, implicating Ω as essential in catalysis across the RS superfamily. Finally, EPR and electron nuclear double resonance spectroscopy establish that Ω involves an Fe-C5' bond between 5'-dAdo· and the [4Fe-4S] cluster. An analogous organometallic bond is found in the well-known adenosylcobalamin (coenzyme B12) cofactor used to initiate radical reactions via a 5'-dAdo· intermediate. Liberation of a reactive 5'-dAdo· intermediate via homolytic metal-carbon bond cleavage thus appears to be similar for Ω and coenzyme B12. However, coenzyme B12 is involved in enzymes catalyzing only a small number (∼12) of distinct reactions, whereas the RS superfamily has more than 100 000 distinct sequences and over 80 reaction types characterized to date. The appearance of Ω across the RS superfamily therefore dramatically enlarges the sphere of bio-organometallic chemistry in Nature.

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

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 (-):CO2H may be generated through an elimination reaction. We tested this hypothesis with both wild type and HydG variants defective in second iron-sulfur cluster coordination by measuring the in vitro production of CO, CN(-), and (-):CO2H-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.

Crystal structure of HydG from Carboxydothermus hydrogenoformans: a trifunctional [FeFe]-hydrogenase maturase.

The structure of the radical S-adenosyl-L-methionine (SAM) [FeFe]-hydrogenase maturase HydG involved in CN(-) /CO synthesis is characterized by two internal tunnels connecting its tyrosine-binding pocket with the external medium and the C-terminal Fe4 S4 cluster-containing region. A comparison with a tryptophan-bound NosL structure suggests that substrate binding causes the closing of the first tunnel and, along with mutagenesis studies, that tyrosine binds to HydG with its amino group well positioned for H-abstraction by SAM. In this orientation the dehydroglycine (DHG) fragment caused by tyrosine Cα-Cß bond scission can readily migrate through the second tunnel towards the C-terminal domain where both CN(-) and CO are synthesized. Our HydG structure appears to be in a relaxed state with its C-terminal cluster CysX2 CysX22 Cys motif exposed to solvent. A rotation of this domain coupled to Fe4 S4 cluster assembly would bury its putatively reactive unique Fe ion thereby allowing it to interact with DHG.

IscS from Archaeoglobus fulgidus has no desulfurase activity but may provide a cysteine ligand for [Fe2S2] cluster assembly.

Iron sulfur ([Fe-S]) clusters are essential prosthetic groups involved in fundamental cell processes such as gene expression regulation, electron transfer and Lewis acid base chemistry. Central components of their biogenesis are pyridoxal-5'-phosphate (PLP) dependent l-cysteine desulfurases, which provide the necessary S atoms for [Fe-S] cluster assembly. The archaeon Archaeoglobus fulgidus (Af) has two ORFs, which although annotated as l-cysteine desulfurases of the ISC type (IscS), lack the essential Lys residue (K199 in Af) that forms a Schiff base with PLP. We have previously determined the structure of an Af(IscU-D35A-IscS)2 complex heterologously expressed in Escherichia coli and found it to contain a [Fe2S2] cluster. In order to understand the origin of sulfide in that structure we have performed a series of functional tests using wild type and mutated forms of AfIscS. In addition, we have determined the crystal structure of an AfIscS-D199K mutant. From these studies we conclude that: i) AfIscS has no desulfurase activity; ii) in our in vitro [Fe2S2] cluster assembly experiments, sulfide ions are non-enzymatically generated by a mixture of iron, l-cysteine and PLP and iii) the physiological role of AfIscS may be to provide a cysteine ligand to the nascent cluster as observed in the [Fe2S2]-Af(IscU-D35A-IscS)2 complex. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.