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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).