A shift between mineral and nonmineral sources of iron and sulfur causes proteome-wide changes in Methanosarcina barkeri.

Iron (Fe) and sulfur (S) are required elements for life, and changes in their availability can limit the ecological distribution and function of microorganisms. In anoxic environments, soluble Fe typically exists as ferrous iron [Fe(II)] and S as sulfide (HS-). These species exhibit a strong affinity that ultimately drives the formation of sedimentary pyrite (FeS2). Recently, paradigm-shifting studies indicate that Fe and S in FeS2 can be made bioavailable by methanogens through a reductive dissolution process. However, the impact of the utilization of FeS2, as opposed to canonical Fe and S sources, on the phenotype of cells is not fully understood. Here, shotgun proteomics was utilized to measure changes in the phenotype of Methanosarcina barkeri MS grown with FeS2, Fe(II)/HS-, or Fe(II)/cysteine. Shotgun proteomics tracked 1,019 proteins overall, with 307 observed to change between growth conditions. Functional characterization and pathway analyses revealed these changes to be systemic and largely tangential to Fe/S metabolism. As a final step, the proteomics data were viewed with respect to previously collected transcriptomics data to deepen the analysis. Presented here is evidence that M. barkeri adopts distinct phenotypes to exploit specific sources of Fe and S in its environment. This is supported by observed protein abundance changes across broad categories of cellular biology. DNA adjacent metabolism, central carbon metabolism methanogenesis, metal trafficking, quorum sensing, and porphyrin biosynthesis pathways are all features in the phenotypic differentiation. Differences in trace metal availability attributed to complexation with HS-, either as a component of the growth medium [Fe(II)/HS-] or generated through reduction of FeS2, were likely a major factor underpinning these phenotypic differences.IMPORTANCEThe methanogenic archaeon Methanosarcina barkeri holds great potential for industrial bio-mining and energy generation technologies. Much of the biochemistry of this microbe is poorly understood, and its characterization will provide a glimpse into biological processes that evolved close to life's origin. The discovery of its ability to extract iron and sulfur from bulk, solid-phase minerals shifted a longstanding paradigm that these elements were inaccessible to biological systems. The full elucidation of this process has the potential to help scientists and engineers extract valuable metals from low-grade ore and mine waste generating energy in the form of methane while doing so.

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.

Proteomic Analysis of Methanococcus voltae Grown in the Presence of Mineral and Nonmineral Sources of Iron and Sulfur.

Iron sulfur (Fe-S) proteins are essential and ubiquitous across all domains of life, yet the mechanisms underpinning assimilation of iron (Fe) and sulfur (S) and biogenesis of Fe-S clusters are poorly understood. This is particularly true for anaerobic methanogenic archaea, which are known to employ more Fe-S proteins than other prokaryotes. Here, we utilized a deep proteomics analysis of Methanococcus voltae A3 cultured in the presence of either synthetic pyrite (FeS2) or aqueous forms of ferrous iron and sulfide to elucidate physiological responses to growth on mineral or nonmineral sources of Fe and S. The liquid chromatography-mass spectrometry (LCMS) shotgun proteomics analysis included 77% of the predicted proteome. Through a comparative analysis of intra- and extracellular proteomes, candidate proteins associated with FeS2 reductive dissolution, Fe and S acquisition, and the subsequent transport, trafficking, and storage of Fe and S were identified. The proteomic response shows a large and balanced change, suggesting that M. voltae makes physiological adjustments involving a range of biochemical processes based on the available nutrient source. Among the proteins differentially regulated were members of core methanogenesis, oxidoreductases, membrane proteins putatively involved in transport, Fe-S binding ferredoxin and radical S-adenosylmethionine proteins, ribosomal proteins, and intracellular proteins involved in Fe-S cluster assembly and storage. This work improves our understanding of ancient biogeochemical processes and can support efforts in biomining of minerals. IMPORTANCE Clusters of iron and sulfur are key components of the active sites of enzymes that facilitate microbial conversion of light or electrical energy into chemical bonds. The proteins responsible for transporting iron and sulfur into cells and assembling these elements into metal clusters are not well understood. Using a microorganism that has an unusually high demand for iron and sulfur, we conducted a global investigation of cellular proteins and how they change based on the mineral forms of iron and sulfur. Understanding this process will answer questions about life on early earth and has application in biomining and sustainable sources of energy.

[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.

The B12-independent glycerol dehydratase activating enzyme from Clostridium butyricum cleaves SAM to produce 5'-deoxyadenosine and not 5'-deoxy-5'-(methylthio)adenosine.

Glycerol dehydratase activating enzyme (GD-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential amino acid backbone radical onto glycerol dehydratase in bacteria under anaerobic conditions. Although GD-AE is closely homologous to other radical SAM activases that have been shown to cleave the S-C(5') bond of SAM to produce 5'-deoxyadenosine (5'-dAdoH) and methionine, GD-AE from Clostridium butyricum has been reported to instead cleave the S-C(γ) bond of SAM to yield 5'-deoxy-5'-(methylthio)adenosine (MTA). Here we re-investigate the SAM cleavage reaction catalyzed by GD-AE and show that it produces the widely observed 5'-dAdoH, and not the less conventional product MTA.

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.

Examining Pathways of Iron and Sulfur Acquisition, Trafficking, Deployment, and Storage in Mineral-Grown Methanogen Cells.

Methanogens have a high demand for iron (Fe) and sulfur (S); however, little is known of how they acquire, deploy, and store these elements and how this, in turn, affects their physiology. Methanogens were recently shown to reduce pyrite (FeS2), generating aqueous iron sulfide (FeSaq) clusters that are likely assimilated as a source of Fe and S. Here, we compared the phenotypes of Methanococcus voltae grown with FeS2 or ferrous iron [Fe(II)] and sulfide (HS-). FeS2-grown cells are 33% smaller yet have 193% more Fe than Fe(II)/HS--grown cells. Whole-cell electron paramagnetic resonance revealed similar distributions of paramagnetic Fe, although FeS2-grown cells showed a broad spectral feature attributed to intracellular thioferrate-like nanoparticles. Differential proteomic analyses showed similar expression of core methanogenesis enzymes, indicating that Fe and S source does not substantively alter the energy metabolism of cells. However, a homolog of the Fe(II) transporter FeoB and its putative transcriptional regulator DtxR were up-expressed in FeS2-grown cells, suggesting that cells sense Fe(II) limitation. Two homologs of IssA, a protein putatively involved in coordinating thioferrate nanoparticles, were also up-expressed in FeS2-grown cells. We interpret these data to indicate that, in FeS2-grown cells, DtxR cannot sense Fe(II) and therefore cannot downregulate FeoB. We suggest this is due to the transport of Fe(II) complexed with sulfide (FeSaq), leading to excess Fe that is sequestered by IssA as a thioferrate-like species. This model provides a framework for the design of targeted experiments aimed at further characterizing Fe acquisition and homeostasis in M. voltae and other methanogens. IMPORTANCE FeS2 is the most abundant sulfide mineral in the Earth's crust and is common in environments inhabited by methanogenic archaea. FeS2 can be reduced by methanogens, yielding aqueous FeSaq clusters that are thought to be a source of Fe and S. Here, we show that growth of Methanococcus voltae on FeS2 results in smaller cell size and higher Fe content per cell, with Fe likely stored intracellularly as thioferrate-like nanoparticles. Fe(II) transporters and storage proteins were upregulated in FeS2-grown cells. These responses are interpreted to result from cells incorrectly sensing Fe(II) limitation due to assimilation of Fe(II) as FeSaq. These findings have implications for our understanding of how Fe/S availability influences methanogen physiology and the biogeochemical cycling of these elements.

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.

Natural selection based on coordination chemistry: computational assessment of [4Fe-4S]-maquettes with non-coded amino acids.

Cysteine is the only coded amino acid in biology that contains a thiol functional group. Deprotonated thiolate is essential for anchoring iron-sulfur ([Fe-S]) clusters, as prosthetic groups to the protein matrix. [Fe-S] metalloproteins and metalloenzymes are involved in biological electron transfer, radical chemistry, small molecule activation and signalling. These are key metabolic and regulatory processes that would likely have been present in the earliest organisms. In the context of emergence of life theories, the selection and evolution of the cysteine-specific R-CH2-SH side chain is a fascinating question to confront. We undertook a computational [4Fe-4S]-maquette modelling approach to evaluate how side chain length can influence [Fe-S] cluster binding and stability in short 7-mer and long 16-mer peptides, which contained either thioglycine, cysteine or homocysteine. Force field-based molecular dynamics simulations for [4Fe-4S] cluster nest formation were supplemented with density functional theory calculations of a ligand-exchange reaction between a preassembled cluster and the peptide. Secondary structure analysis revealed that peptides with cysteine are found with greater frequency nested to bind preformed [4Fe-4S] clusters. Additionally, the presence of the single methylene group in cysteine ligands mitigates the steric bulk, maintains the H-bonding and dipole network, and provides covalent Fe-S(thiolate) bonds that together create the optimal electronic and geometric structural conditions for [4Fe-4S] cluster binding compared to thioglycine or homocysteine ligands. Our theoretical work forms an experimentally testable hypothesis of the natural selection of cysteine through coordination chemistry.

Radical S-adenosylmethionine maquette chemistry: Cx3Cx2C peptide coordinated redox active [4Fe-4S] clusters.

The synthesis and characterization of short peptide-based maquettes of metalloprotein active sites facilitate an inquiry into their structure/function relationships and evolution. The [4Fe-4S]-maquettes of bacterial ferredoxin metalloproteins (Fd) have been used in the past to engineer redox active centers into artificial metalloenzymes. The novelty of our study is the application of maquettes to the superfamily of [4Fe-4S] cluster and S-adenosylmethionine-dependent radical metalloenzymes (radical SAM). The radical SAM superfamily enzymes contain site-differentiated, redox active [4Fe-4S] clusters coordinated to Cx3Cx2C or related motifs, which is in contrast to the Cx2Cx2C motif found in bacterial ferredoxins (Fd). Under an optimized set of experimental conditions, a high degree of reconstitution (80-100%) was achieved for both radical SAM- and Fd-maquettes. Negligible chemical speciation was observed for all sequences, with predominantly [4Fe-4S]2+ for the 'as-reconstituted' state. However, the reduction of [4Fe-4S]2+-maquettes provides low conversion (7-17%) to the paramagnetic [4Fe-4S]+ state, independent of either the spacing of the cysteine residues (Cx3Cx2C vs. Cx2Cx2C), the nature of intervening amino acids, or the length of the cluster binding motif. In the absence of the stabilizing protein environment, the reduction process is proposed to proceed via [4Fe-4S]2+ cluster disassembly and reassembly in a more reduced state. UV-Vis and EPR spectroscopic techniques are employed as analytical tools to quantitate the as-reconstituted (or oxidized) and one-electron reduced states of the [4Fe-4S] clusters, respectively. We demonstrate that short Fd and radical SAM derived 7- to 9-mer peptides containing appropriate cysteine motifs function equally well in coordinating redox active [4Fe-4S] clusters.

H-cluster assembly intermediates built on HydF by the radical SAM enzymes HydE and HydG.

[FeFe]-hydrogenase catalyzes the reversible reduction of protons to H2 at a complex metallocofactor site, the H-cluster. Biosynthesis of this active-site H-cluster requires three maturation enzymes: the radical S-adenosylmethionine enzymes HydE and HydG synthesize the nonprotein ligands, while the GTPase HydF provides a scaffold for assembly of the 2Fe subcluster of the H-cluster ([2Fe]H) prior to its transfer to hydrogenase. To delineate the assembly and delivery steps for the 2Fe precursor cluster coordinated to HydF ([2Fe]F), we have heterologously expressed HydF in the presence of HydE alone (HydFE) or HydG alone (HydFG), and characterized the resulting purified HydFE and HydFG using UV-visible, EPR, and FTIR spectroscopies and biochemical assays. The iron-sulfur clusters on HydF are modified by co-expression with HydE or HydG, as evidenced by the changes in the visible, EPR, and FTIR spectral features. Further, biochemical assays show that HydFE is capable of activating HydAΔEFG to a limited extent (~ 1% of WT) even though the normal source of CO and CN- ligands of [2Fe]H (HydG) was absent. Activation assays performed with HydFG, in contrast, exhibit no ability to mature HydAΔEFG. It appears that in the case of HydFE, trace diatomics from the cellular environment are incorporated into a [2Fe]F-like precursor on HydF in the absence of HydG. We conclude that the product of HydE, presumably the dithiomethylamine ligand of [2Fe]H, is absolutely essential to the activation process, while the diatomic products of HydG can be provided from alternate sources.

Photoinduced Electron Transfer in a Radical SAM Enzyme Generates an S-Adenosylmethionine Derived Methyl Radical.

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 ΩM. 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•.

Characterization of the Preprocessed Copper Site Equilibrium in Amine Oxidase and Assignment of the Reactive Copper Site in Topaquinone Biogenesis.

Copper-dependent amine oxidases produce their redox active cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), via the CuII-catalyzed oxygenation of an active site tyrosine. This study addresses possible mechanisms for this biogenesis process by presenting the geometric and electronic structure characterization of the CuII-bound, prebiogenesis (preprocessed) active site of the enzyme Arthrobacter globiformis amine oxidase (AGAO). CuII-loading into the preprocessed AGAO active site is slow ( kobs = 0.13 h-1), and is preceded by CuII binding in a separate kinetically favored site that is distinct from the active site. Preprocessed active site CuII is in a thermal equilibrium between two species, an entropically favored form with tyrosine protonated and unbound from the CuII site, and an enthalpically favored form with tyrosine bound deprotonated to the CuII active site. It is shown that the CuII-tyrosinate bound form is directly active in biogenesis. The electronic structure determined for the reactive form of the preprocessed CuII active site is inconsistent with a biogenesis pathway that proceeds through a CuI-tyrosyl radical intermediate, but consistent with a pathway that overcomes the spin forbidden reaction of 3O2 with the bound singlet substrate via a three-electron concerted charge-transfer mechanism.

Secondary structure analysis of peptides with relevance to iron-sulfur cluster nesting.

Peptides coordinated to iron-sulfur clusters, referred to as maquettes, represent a synthetic strategy for constructing biomimetic models of iron-sulfur metalloproteins. These maquettes have been successfully employed as building blocks of engineered heme-containing proteins with electron-transfer functionality; however, they have yet to be explored in reactivity studies. The concept of iron-sulfur nesting in peptides is a leading hypothesis in Origins-of-Life research as a plausible path to bridge the discontinuity between prebiotic chemical transformations and extant enzyme catalysis. Based on past biomimetic and biochemical research, we put forward a mechanism of maquette reconstitution that guides our development of computational tools and methodologies. In this study, we examined a key feature of the first stage of maquette formation, which is the secondary structure of aqueous peptide models using molecular dynamics simulations based on the AMBER99SB empirical force field. We compared and contrasted S…S distances, [2Fe-2S] and [4Fe-4S] nests, and peptide conformations via Ramachandran plots for dissolved Cys and Gly amino acids, the CGGCGGC 7-mer, and the GGCGGGCGGCGGW 16-mer peptide. Analytical tools were developed for following the evolution of secondary structural features related to [Fe-S] cluster nesting along 100 ns trajectories. Simulations demonstrated the omnipresence of peptide nests for preformed [2Fe-2S] clusters; however, [4Fe-4S] cluster nests were observed only for the 16-mer peptide with lifetimes of a few nanoseconds. The origin of the [4Fe-4S] nest and its stability was linked to a "kinked-ribbon" peptide conformation. Our computational approach lays the foundation for transitioning into subsequent stages of maquette reconstitution, those being the formation of iron ion/iron-sulfur coordinated peptides. © 2018 Wiley Periodicals, Inc.

Compositional and structural insights into the nature of the H-cluster precursor on HydF.

Assembly of an active [FeFe]-hydrogenase requires dedicated maturation enzymes that generate the active-site H-cluster: the radical SAM enzymes HydE and HydG synthesize the unusual non-protein ligands - carbon monoxide, cyanide, and dithiomethylamine - while the GTPase HydF serves as a scaffold for assembly of the 2Fe subcluster containing these ligands. In the current study, enzymatically cluster-loaded HydF ([2Fe]F) is produced by co-expression with HydE and HydG in an Escherichia coli host followed by isolation and examination by FTIR and EPR spectroscopy. FTIR reveals the presence of well-defined terminal CO and CN- ligands; however, unlike in the [FeFe]-hydrogenase, no bridging CO is observed. Exposure of this loaded HydF to exogenous CO or H2 produces no significant changes to the FTIR spectrum, indicating that, unlike in the [FeFe]-hydrogenase, the 2Fe cluster in loaded HydF is coordinatively saturated and relatively unreactive. EPR spectroscopy reveals the presence of both [4Fe-4S] and [2Fe-2S] clusters on this loaded HydF, but provides no direct evidence for these being linked to the [2Fe]F. Using the chemical reactivity and FTIR data, a large collection of computational models were evaluated. Their scaled quantum chemical vibrational spectra allowed us to score various [2Fe]F structures in terms of their ability to reproduce the diatomic stretching frequencies observed in the FTIR experimental spectra. Collectively, the results provide new insights that support the presence of a diamagnetic, but spin-polarized FeI-FeI oxidation state for the [2Fe]F precursor cluster that is coordinated by 4 CO and 2 CN- ligands, and bridged to an adjacent iron-sulfur cluster through one of the CN- ligands.

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.

Decarboxylation involving a ferryl, propionate, and a tyrosyl group in a radical relay yields heme b.

The H2O2-dependent oxidative decarboxylation of coproheme III is the final step in the biosynthesis of heme b in many microbes. However, the coproheme decarboxylase reaction mechanism is unclear. The structure of the decarboxylase in complex with coproheme III suggested that the substrate iron, reactive propionates, and an active-site tyrosine convey a net 2e-/2H+ from each propionate to an activated form of H2O2 Time-resolved EPR spectroscopy revealed that Tyr-145 formed a radical species within 30 s of the reaction of the enzyme-coproheme complex with H2O2 This radical disappeared over the next 270 s, consistent with a catalytic intermediate. Use of the harderoheme III intermediate as substrate or substitutions of redox-active side chains (W198F, W157F, or Y113S) did not strongly affect the appearance or intensity of the radical spectrum measured 30 s after initiating the reaction with H2O2, nor did it change the ∼270 s required for the radical signal to recede to ≤10% of its initial intensity. These results suggested Tyr-145 as the site of a catalytic radical involved in decarboxylating both propionates. Tyr-145• was accompanied by partial loss of the initially present Fe(III) EPR signal intensity, consistent with the possible formation of Fe(IV)=O. Site-specifically deuterated coproheme gave rise to a kinetic isotope effect of ∼2 on the decarboxylation rate constant, indicating that cleavage of the propionate Cß-H bond was partly rate-limiting. The inferred mechanism requires two consecutive hydrogen atom transfers, first from Tyr-145 to the substrate Fe/H2O2 intermediate and then from the propionate Cß-H to Tyr-145•.