Cysteine as a ligand platform in the biosynthesis of the FeFe hydrogenase H cluster.

Hydrogenases catalyze the redox interconversion of protons and H2, an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [4Fe-4S]H subcluster coupled to a [2Fe]H subcluster bound by CO, CN(-), and azadithiolate ligands. The [2Fe]H subcluster is assembled by the maturases HydE, HydF, and HydG. HydG is a member of the radical S-adenosyl-L-methionine family of enzymes that transforms Fe and L-tyrosine into an [Fe(CO)2(CN)] synthon that is incorporated into the H cluster. Although it is thought that the site of synthon formation in HydG is the "dangler" Fe of a [5Fe] cluster, many mechanistic aspects of this chemistry remain unresolved including the full ligand set of the synthon, how the dangler Fe initially binds to HydG, and how the synthon is released at the end of the reaction. To address these questions, we herein show that L-cysteine (Cys) binds the auxiliary [4Fe-4S] cluster of HydG and further chelates the dangler Fe. We also demonstrate that a [4Fe-4S]aux[CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO)2(CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Thus, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe(2+) binding and synthon release.

Biosynthesis of the [FeFe] Hydrogenase H Cluster: A Central Role for the Radical SAM Enzyme HydG.

Hydrogenase enzymes catalyze the rapid and reversible interconversion of H2 with protons and electrons. The active site of the [FeFe] hydrogenase is the H cluster, which consists of a [4Fe-4S]H subcluster linked to an organometallic [2Fe]H subcluster. Understanding the biosynthesis and catalytic mechanism of this structurally unusual active site will aid in the development of synthetic and biological hydrogenase catalysts for applications in solar fuel generation. The [2Fe]H subcluster is synthesized and inserted by three maturase enzymes-HydE, HydF, and HydG-in a complex process that involves inorganic, organometallic, and organic radical chemistry. HydG is a member of the radical S-adenosyl-l-methionine (SAM) family of enzymes and is thought to play a prominent role in [2Fe]H subcluster biosynthesis by converting inorganic Fe(2+), l-cysteine (Cys), and l-tyrosine (Tyr) into an organometallic [(Cys)Fe(CO)2(CN)](-) intermediate that is eventually incorporated into the [2Fe]H subcluster. In this Forum Article, the mechanism of [2Fe]H subcluster biosynthesis is discussed with a focus on how this key [(Cys)Fe(CO)2(CN)](-) species is formed. Particular attention is given to the initial metallocluster composition of HydG, the modes of substrate binding (Fe(2+), Cys, Tyr, and SAM), the mechanism of SAM-mediated Tyr cleavage to CO and CN(-), and the identification of the final organometallic products of the reaction.

The cyanide ligands of [FeFe] hydrogenase: pulse EPR studies of (13)C and (15)N-labeled H-cluster.

The two cyanide ligands in the assembled cluster of [FeFe] hydrogenase originate from exogenous l-tyrosine. Using selectively labeled tyrosine substrates, the cyanides were isotopically labeled via a recently developed in vitro maturation procedure allowing advanced electron paramagnetic resonance techniques to probe the electronic structure of the catalytic core of the enzyme. The ratio of the isotropic (13)C hyperfine interactions for the two CN(-) ligands-a reporter of spin density on their respective coordinating iron ions-collapses from ≈5.8 for the Hox form of hydrogenase to <2 for the CO-inhibited form. Additionally, when the maturation was carried out using [(15)N]-tyrosine, no features previously ascribed to the nitrogen of the bridging dithiolate ligand were observed suggesting that this bridge is not sourced from tyrosine.

Engineering Clostridium acetobutylicum for production of kerosene and diesel blendstock precursors.

Processes for the biotechnological production of kerosene and diesel blendstocks are often economically unattractive due to low yields and product titers. Recently, Clostridium acetobutylicum fermentation products acetone, butanol, and ethanol (ABE) were shown to serve as precursors for catalytic upgrading to higher chain-length molecules that can be used as fuel substitutes. To produce suitable kerosene and diesel blendstocks, the butanol:acetone ratio of fermentation products needs to be increased to 2-2.5:1, while ethanol production is minimized. Here we show that the overexpression of selected proteins changes the ratio of ABE products relative to the wild type ATCC 824 strain. Overexpression of the native alcohol/aldehyde dehydrogenase (AAD) has been reported to primarily increase ethanol formation in C. acetobutylicum. We found that overexpression of the AAD(D485G) variant increased ethanol titers by 294%. Catalytic upgrading of the 824(aad(D485G)) ABE products resulted in a blend with nearly 50wt%≤C9 products, which are unsuitable for diesel. To selectively increase butanol production, C. beijerinckii aldehyde dehydrogenase and C. ljungdhalii butanol dehydrogenase were co-expressed (strain designate 824(Cb ald-Cl bdh)), which increased butanol titers by 27% to 16.9gL(-1) while acetone and ethanol titers remained essentially unaffected. The solvent ratio from 824(Cb ald-Cl bdh) resulted in more than 80wt% of catalysis products having a carbon chain length≥C11 which amounts to 9.8gL(-1) of products suitable as kerosene or diesel blendstock based on fermentation volume. To further increase solvent production, we investigated expression of both native and heterologous chaperones in C. acetobutylicum. Expression of a heat shock protein (HSP33) from Bacillus psychrosaccharolyticus increased the total solvent titer by 22%. Co-expression of HSP33 and aldehyde/butanol dehydrogenases further increased ABE formation as well as acetone and butanol yields. HSP33 was identified as the first heterologous chaperone that significantly increases solvent titers above wild type C. acetobutylicum levels, which can be combined with metabolic engineering to further increase solvent production.

Nuclear resonance vibrational spectroscopy and electron paramagnetic resonance spectroscopy of 57Fe-enriched [FeFe] hydrogenase indicate stepwise assembly of the H-cluster.

The [FeFe] hydrogenase from Clostridium pasteurianum (CpI) harbors four Fe-S clusters that facilitate the transfer of an electron to the H-cluster, a ligand-coordinated six-iron prosthetic group that catalyzes the redox interconversion of protons and H(2). Here, we have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the iron centers in CpI, and we compare our data to that for a [4Fe-4S] ferredoxin as well as a model complex resembling the [2Fe](H) catalytic domain of the H-cluster. To enrich the hydrogenase with (57)Fe nuclei, we used cell-free methods to post-translationally mature the enzyme. Specifically, inactive CpI apoprotein with (56)Fe-labeled Fe-S clusters was activated in vitro using (57)Fe-enriched maturation proteins. This approach enabled us to selectively label the [2Fe](H) subcluster with (57)Fe, which NRVS confirms by detecting (57)Fe-CO and (57)Fe-CN normal modes from the H-cluster nonprotein ligands. The NRVS and iron quantification results also suggest that the hydrogenase contains a second (57)Fe-S cluster. Electron paramagnetic resonance (EPR) spectroscopy indicates that this (57)Fe-enriched metal center is not the [4Fe-4S](H) subcluster of the H-cluster. This finding demonstrates that the CpI hydrogenase retained an (56)Fe-enriched [4Fe-4S](H) cluster during in vitro maturation, providing unambiguous evidence of stepwise assembly of the H-cluster. In addition, this work represents the first NRVS characterization of [FeFe] hydrogenases.

Cell-free synthesis and maturation of [FeFe] hydrogenases.

[FeFe] hydrogenases catalyze the reversible reduction of protons to molecular hydrogen (Adams (1990); Biochim Biophys Acta 1020(2): 115-145) and are of significant interest for the biological production of hydrogen fuel. They are complex proteins with active sites containing iron, sulfur, and carbon monoxide and cyanide ligands (Peters et al. (1998); Science 282(5395): 1853-1858). Maturation enzymes for [FeFe] hydrogenases have been identified (Posewitz et al. (2004); J Biol Chem 279(24): 25711-25720), but complete mechanisms have not yet been elucidated. The study of [FeFe] hydrogenases has been impeded by the lack of an easily manipulated expression/activation system capable of producing these complex and extremely oxygen-sensitive enzymes. Here we show the first expression of functional [FeFe] hydrogenases in an Escherichia coli-based cell-free transcription/translation system. We have produced and matured both algal and bacterial hydrogenases using E. coli cell extracts containing the HydG, HydE, and HydF proteins from Shewanella oneidensis. The current system produces approximately 22 microg/mL of active protein, constituting approximately 44% of the total protein produced. Active protein yield is greatly enhanced by pre-incubation of the maturation enzyme-containing extract with inorganic iron and sulfur for reconstitution of the [Fe-S] clusters in HydG, HydE, and HydF. The absence of cell walls permits direct addition of cofactors and substrates, enabling rapid production of active protein and providing control over the maturation conditions. These new capabilities will enhance the investigation of complex proteins requiring helper proteins for maturation and move us closer to the development of improved hydrogenases for biological production of hydrogen as a clean, renewable alternative fuel.

Cell-free synthesis of the H-cluster: a model for the in vitro assembly of metalloprotein metal centers.

Many organometallic cofactors are highly complex and require multiple accessory proteins for both their assembly and transfer to a target protein. A cell-free system in which the biosynthetic pathway for a prosthetic group has been fully or even partially reconstructed enables investigations of the reaction sequence as well as the cofactor itself. As a model for the in vitro assembly of protein-bound metal centers, we describe a procedure for the cell-free synthesis of the H-cluster in the context of producing purified and active [FeFe] hydrogenase samples for spectroscopic studies. In general terms, this in vitro system is a combination of non-purified accessory proteins, exogenous substrates, and purified hydrogenase apoprotein. We also describe methods for making the required components used in the cell-free system. Specifically, these procedures include anaerobic expression of heterologous metalloproteins in Escherichia coli, anaerobic cell lysate production, and anaerobic metalloprotein purification using Strep-Tactin(®) chromatography.

The HydG enzyme generates an Fe(CO)2(CN) synthon in assembly of the FeFe hydrogenase H-cluster.

Three iron-sulfur proteins--HydE, HydF, and HydG--play a key role in the synthesis of the [2Fe](H) component of the catalytic H-cluster of FeFe hydrogenase. The radical S-adenosyl-L-methionine enzyme HydG lyses free tyrosine to produce p-cresol and the CO and CN(-) ligands of the [2Fe](H) cluster. Here, we applied stopped-flow Fourier transform infrared and electron-nuclear double resonance spectroscopies to probe the formation of HydG-bound Fe-containing species bearing CO and CN(-) ligands with spectroscopic signatures that evolve on the 1- to 1000-second time scale. Through study of the (13)C, (15)N, and (57)Fe isotopologs of these intermediates and products, we identify the final HydG-bound species as an organometallic Fe(CO)2(CN) synthon that is ultimately transferred to apohydrogenase to form the [2Fe](H) component of the H-cluster.

A radical intermediate in tyrosine scission to the CO and CN- ligands of FeFe hydrogenase.

The radical S-adenosylmethionine (SAM) enzyme HydG lyses free l-tyrosine to produce CO and CN(-) for the assembly of the catalytic H cluster of FeFe hydrogenase. We used electron paramagnetic resonance spectroscopy to detect and characterize HydG reaction intermediates generated with a set of (2)H, (13)C, and (15)N nuclear spin-labeled tyrosine substrates. We propose a detailed reaction mechanism in which the radical SAM reaction, initiated at an N-terminal 4Fe-4S cluster, generates a tyrosine radical bound to a C-terminal 4Fe-4S cluster. Heterolytic cleavage of this tyrosine radical at the Cα-Cß bond forms a transient 4-oxidobenzyl (4OB(•)) radical and a dehydroglycine bound to the C-terminal 4Fe-4S cluster. Electron and proton transfer to this 4OB(•) radical forms p-cresol, with the conversion of this dehydroglycine ligand to Fe-bound CO and CN(-), a key intermediate in the assembly of the 2Fe subunit of the H cluster.

New insights into [FeFe] hydrogenase activation and maturase function.

[FeFe] hydrogenases catalyze H(2) production using the H-cluster, an iron-sulfur cofactor that contains carbon monoxide (CO), cyanide (CN(-)), and a dithiolate bridging ligand. The HydE, HydF, and HydG maturases assist in assembling the H-cluster and maturing hydrogenases into their catalytically active form. Characterization of these maturases and in vitro hydrogenase activation methods have helped elucidate steps in the H-cluster biosynthetic pathway such as the HydG-catalyzed generation of the CO and CN(-) ligands from free tyrosine. We have refined our cell-free approach for H-cluster synthesis and hydrogenase maturation by using separately expressed and purified HydE, HydF, and HydG. In this report, we illustrate how substrates and protein constituents influence hydrogenase activation, and for the first time, we show that each maturase can function catalytically during the maturation process. With precise control over the biomolecular components, we also provide evidence for H-cluster synthesis in the absence of either HydE or HydF, and we further show that hydrogenase activation can occur without exogenous tyrosine. Given these findings, we suggest a new reaction sequence for the [FeFe] hydrogenase maturation pathway. In our model, HydG independently synthesizes an iron-based compound with CO and CN(-) ligands that is a precursor to the H-cluster [2Fe](H) subunit, and which we have termed HydG-co. We further propose that HydF is a transferase that stabilizes HydG-co and also shuttles the complete [2Fe](H) subcluster to the hydrogenase, a translocation process that may be catalyzed by HydE. In summary, this report describes the first example of reconstructing the [FeFe] hydrogenase maturation pathway using purified maturases and subsequently utilizing this in vitro system to better understand the roles of HydE, HydF, and HydG.

Cell-free H-cluster synthesis and [FeFe] hydrogenase activation: all five CO and CN⁻ ligands derive from tyrosine.

[FeFe] hydrogenases are promising catalysts for producing hydrogen as a sustainable fuel and chemical feedstock, and they also serve as paradigms for biomimetic hydrogen-evolving compounds. Hydrogen formation is catalyzed by the H-cluster, a unique iron-based cofactor requiring three carbon monoxide (CO) and two cyanide (CN⁻) ligands as well as a dithiolate bridge. Three accessory proteins (HydE, HydF, and HydG) are presumably responsible for assembling and installing the H-cluster, yet their precise roles and the biosynthetic pathway have yet to be fully defined. In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation. Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN⁻ ligands derive respectively from the carboxylate and amino substituents of tyrosine. Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the protein-protein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation.

High-yield expression of heterologous [FeFe] hydrogenases in Escherichia coli.

BACKGROUND: The realization of hydrogenase-based technologies for renewable H(2) production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases. PRINCIPAL FINDINGS: In this report, we describe an improved Escherichia coli-based expression system capable of producing 8-30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H(2) evolution with rates comparable to those of enzymes isolated from their respective native organisms. SIGNIFICANCE: The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H(2)-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.

Assembly of erodible, DNA-containing thin films on the surfaces of polymer microparticles: toward a layer-by-layer approach to the delivery of DNA to antigen-presenting cells.

We report a layer-by-layer approach to the assembly of ultrathin and erodible DNA-containing films on the surfaces of polymer microparticles. DNA-containing multilayered films were fabricated layer-by-layer on the surfaces of polystyrene microspheres (approximately 6 microm) by iterative and alternating cycles of particle suspension, centrifugation and resuspension in solutions of plasmid DNA and a hydrolytically degradable polyamine. Film growth occurred in a stepwise manner, as demonstrated by characterization of the zeta potentials and fluorescence intensities of film-coated particles during film assembly. Characterization of film-coated particles by confocal fluorescence microscopy and scanning electron microscopy revealed the multilayered particle coatings to be smooth, uniform and free of large-scale physical defects. Film-coated microparticles sustained the release of transcriptionally active DNA into solution for approximately three days when incubated in physiologically relevant media. Previous studies have demonstrated that the adsorption of DNA onto the surfaces of cationic microparticles can be used to target the delivery of DNA to antigen-presenting cells. As a first step toward the application of this layer-by-layer approach to the development of methods for the delivery of DNA to antigen-presenting cells, we demonstrated that film-coated microparticles could be used to transport DNA into macrophage cells in vitro using a model mouse macrophage cell line. Our results suggest the basis of a general approach that could, with further development, prove useful for the delivery of DNA-encoded antigens to macrophages, or other antigen-presenting cells, and provide new materials-based methods for the formulation and delivery of DNA vaccines.

Tyrosine, cysteine, and S-adenosyl methionine stimulate in vitro [FeFe] hydrogenase activation.

BACKGROUND: [FeFe] hydrogenases are metalloenzymes involved in the anaerobic metabolism of H(2). These proteins are distinguished by an active site cofactor known as the H-cluster. This unique [6Fe-6S] complex contains multiple non-protein moieties and requires several maturation enzymes for its assembly. The pathways and biochemical precursors for H-cluster biosynthesis have yet to be elucidated. PRINCIPAL FINDINGS: We report an in vitro maturation system in which, for the first time, chemical additives enhance [FeFe] hydrogenase activation, thus signifying in situ H-cluster biosynthesis. The maturation system is comprised of purified hydrogenase apoprotein; a dialyzed Escherichia coli cell lysate containing heterologous HydE, HydF, and HydG maturases; and exogenous small molecules. Following anaerobic incubation of the Chlamydomonas reinhardtii HydA1 apohydrogenase with S-adenosyl methionine (SAM), cysteine, tyrosine, iron, sulfide, and the non-purified maturases, hydrogenase activity increased 5-fold relative to incubations without the exogenous substrates. No conditions were identified in which addition of guanosine triphosphate (GTP) improved hydrogenase maturation. SIGNIFICANCE: The in vitro system allows for direct investigation of [FeFe] hydrogenase activation. This work also provides a foundation for studying the biosynthetic mechanisms of H-cluster biosynthesis using solely purified enzymes and chemical additives.