Eric M. Shepard

The organometallic H-cluster of the [FeFe]-hydrogenase is assembled in vivo through a complex process requiring the action of three dedicated maturation enzymes, HydG, HydE, and HydF, as well as the aminomethyl-lipoyl-H-protein (H_met) of the glycine cleavage system (GCS). Here we probe the role of HydF and its [4Fe-4S] cluster in [FeFe]-hydrogenase maturation by using a defined semisynthetic approach in which [Fe^I₂(μ-SH)₂(CO)₄(CN)₂]^2- ([2Fe]_E) is used to bypass HydE and HydG, and GCS components are used in place of cell lysate. We show that inclusion of the iron-sulfur carrier protein NfuA and the high-CO-affinity myoglobin variant Mb^H64L provides dramatically improved hydrogenase activities up to 828 μmol/min/mg, equivalent to the best reported activities for Chlamydomonas reinhardtii [FeFe]-hydrogenase isolated from the native organism. Apo-HydF lacking a [4Fe-4S] cluster provides very little hydrogenase activity; however, full maturation is restored with the addition of NfuA, which we demonstrate reconstitutes the [4Fe-4S] cluster of HydF. In addition, a HydF variant lacking a [4Fe-4S] cluster by changing two cysteine ligands to alanine is completely unable to support either semisynthetic maturation using [2Fe]_E, or full maturation using HydG and HydE, even in the presence of NfuA, demonstrating that the HydF [4Fe-4S] cluster is absolutely essential for [FeFe]-hydrogenase maturation. The possibility that the HydF [4Fe-4S] cluster plays a role in direct binding of [2Fe]_E is negated by our results with the HydF^D311C variant, which demonstrate that the labile Asp311 cluster ligand is not essential for [2Fe]_E binding and HydA maturation. We therefore conclude that [2Fe]_E binds HydF adjacent to, but not directly coordinated to, the [4Fe-4S] cluster. The HydF [4Fe-4S] cluster is proposed to be essential due to its impact on the [2Fe]_E binding orientation and the ability of the HydF/[2Fe]_E complex to form productive interactions with H_met or the H_met/T-protein complex during DTMA ligand biosynthesis.

The [FeFe]-hydrogenase can be matured using a defined semisynthetic approach in which the synthetic complex [2Fe]_Ebypasses the need for HydE and HydG, and glycine cleavage system components synthesize DTMA on [2Fe]_Ewhile it is bound to HydF.

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.

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.

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.

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.

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.