Stella Impano

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.

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^.. 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 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 77 K, the ethyl radical adds to the [4Fe‐4S]²⁺ cluster, generating an ethyl‐[4Fe‐4S]³⁺ organometallic species termed Ω_E.

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^.. 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 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 77 K, the ethyl radical adds to the [4Fe‐4S]²⁺ cluster, generating an ethyl‐[4Fe‐4S]³⁺ organometallic species termed Ω_E.

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.

Radical SAM (RS) enzymes use S-adenosyl-l-methionine (SAM) and a [4Fe–4S] cluster to initiate a broad spectrum of radical transformations throughout all kingdoms of life. We report here that low-temperature photoinduced electron transfer from the [4Fe–4S]¹⁺ cluster to bound SAM in the active site of the hydrogenase maturase RS enzyme, HydG, results in specific homolytic cleavage of the S–CH₃ bond of SAM, rather than the S–C5′ bond as in the enzyme-catalyzed (thermal) HydG reaction. This result is in stark contrast to a recent report in which photoinduced ET in the RS enzyme pyruvate formate-lyase activating enzyme cleaved the S–C5′ bond to generate a 5′-deoxyadenosyl radical, and provides the first direct evidence for homolytic S–CH₃ bond cleavage in a RS enzyme. Photoinduced ET in HydG generates a trapped ^•CH₃ radical, as well as a small population of an organometallic species with an Fe–CH₃ bond, denoted Ω_M. The ^•CH₃ radical is surprisingly found to exhibit rotational diffusion in the HydG active site at temperatures as low as 40 K, and is rapidly quenched: whereas 5′-dAdo^• is stable indefinitely at 77 K, ^•CH₃ quenches with a half-time of ∼2 min at this temperature. The rapid quenching and rotational/translational freedom of ^•CH₃ shows that enzymes would be unable to harness this radical as a regio- and stereospecific H atom abstractor during catalysis, in contrast to the exquisite control achieved with the enzymatically generated 5′-dAdo^•.

The 5′-deoxyadenosyl radical (5′-dAdo·) abstracts a substrate H atom as the first step in radical-based transformations catalyzed by adenosylcobalamin-dependent and radical S-adenosyl-l-methionine (RS) enzymes. Notwithstanding its central biological role, 5′-dAdo· has eluded characterization despite efforts spanning more than a half-century. Here, we report generation of 5′-dAdo· in a RS enzyme active site at 12 K using a novel approach involving cryogenic photoinduced electron transfer from the [4Fe–4S]⁺ cluster to the coordinated S-adenosylmethionine (SAM) to induce homolytic S–C5′ bond cleavage. We unequivocally reveal the structure of this long-sought radical species through the use of electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies with isotopic labeling, complemented by density-functional computations: a planar C5′ (2pπ) radical (∼70% spin occupancy); the C5′(H)₂ plane is rotated by ∼37° (experiment)/39° (DFT) relative to the C5′–C4′–(C4′–H) plane, placing a C5′–H antiperiplanar to the ribose-ring oxygen, which helps stabilize the radical against elimination of the 4′−H. The agreement between φ from experiment and in vacuo DFT indicates that the conformation is intrinsic to 5-dAdo· itself, and not determined by its environment.