Tyrosine, Cysteine, and S-Adenosyl Methionine Stimulate In Vitro [FeFe] Hydrogenase Activation

Background: [FeFe] hydrogenases are metalloenzymes involved in the anaerobic metabolism of H2. 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

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

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

Effects of excluding protein constituents on <i>in vitro</i> CpI activation.

<p>Except for the protein constituents indicated under <i>Condition</i> (Δ), reaction mixtures contained a Δ<i>iscR E. coli</i> lysate (either clarified or desalted), 5 µM HydE, 5 µM HydF, 50 µM HydG, 1 µM CpI, and the standard set of extrinsic substrates. Either no NTP or ATP (15 mM) was added instead of GTP (15 mM) for selected conditions.</p>†<p>CpI specific activities are shown in units of µmol H₂ consumed·min^–1 mg CpI^–1 (n = 4–8).</p

<i>In vitro</i> activation of <i>C. reinhardtii</i> HydA1 and the effects of exogenous small molecules.

<p>2 µM of HydA1 apoprotein was anaerobically incubated with 50–60% vol·vol⁻¹ maturase extract. Exogenous substrates assessed included Fe⁺² (1 mM), S⁻² (1 mM), SAM (2 mM), and 20 aa (2 mM of each amino acid). (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007565#pone-0007565-g002" target="_blank"><i>Fig. 2A</i></a>) When included in reaction mixtures, Fe⁺² and S⁻² were added to maturase extracts 2 hr before addition of apoHydA1 (black bars) or apoHydA1^recon (red bars). When SAM and 20 aa were included, maturase extracts were incubated with these chemical additives for 1 hr prior to HydA1 addition. Final hydrogenase activities determined after 9 hr of incubation are from n = 2 to 5 independent determinations ± SEM. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007565#pone-0007565-g002" target="_blank"><i>Fig. 2B</i></a>) Maturase extracts were reconstituted with Fe⁺² and S⁻² for 2 hr (•,▪,▴) or 0 hr (+) before apoHydA1 addition; extracts were also pre-treated with SAM and 20 aa for 1 hr (▴) or 0 hr (▪,•,+) before adding HydA1 apoprotein (as-isolated: •,▴,+; reconstituted: ▪). Data are from n = 2 independent measurements, and standard errors were less than 10% for all data.</p

EPR spectroscopy of the <i>Shewanella oneidensis</i> HydF maturase.

<p>Continuous-wave EPR measurements were obtained at an X-band frequency (9.39 GHz). Spectra for as-isolated HydF (1.2 mM, solid lines) were measured at 20 K (50 µW), 40 K (0.5 mW), 80 K (5 mW) and 120 K (50 mW). Spectra for DTH-reduced HydF (1.1 mM, dashed lines) were measured at 20 K (200 µW), 40 K (2 mW), 80 K (50 mW) and 120 K (50 mW). The <i>g</i>-values for each signal are indicated in the 20 K spectra. Intensities were normalized to the numbers of scans, temperature, receiver gain, conversion time, modulation amplitude, and square root of microwave power. All spectra are presented without magnification of the normalized intensities. Spectra identical to those for reduced HydF were observed at 20 K and 40 K for the HydF maturase reconstituted <i>in vitro</i> with Fe²⁺, S^2–, cysteine, PLP, and an <i>E. coli</i> lysate for 60 min prior to DTH reduction (0.80 spins per protein).</p

Effects of SAM on <i>in vitro</i> HydA1 maturation.

<p>Maturase extracts were reconstituted with Fe⁺² and S⁻² for 60 min, and then pre-treated for 60 min with the indicated small molecules prior to apoHydA1 addition (3.6–4.6 µM). Reactions mixtures contained 50–70% vol·vol⁻¹ maturase extract. Final concentrations of chemical additives were 1 mM Fe⁺², 1 mM S⁻², 2 mM of each amino acid (20 aa), 2 mM SAM, 1 mM NADPH, and 2 mM SAH. Hydrogenase activities were measured after 8–9 hr of anaerobic incubation. Data are the average for n = 2 to 4 independent determinations ± SEM.</p

Characterization of purified <i>C. reinhardtii</i> HydA1 apohydrogenase.

<p>(<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007565#pone-0007565-g001" target="_blank"><i>Fig. 1A</i></a>) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of pooled elution fractions containing N-his₆-HydA1 apoprotein (48.4 kDa) following aerobic expression in <i>E. coli</i> and subsequent Ni⁺²-affinity chromatography. The molecular weight marker (MWM) is the Mark 12™ protein ladder (Invitrogen). Intermediate lanes of the SDS-polyacrylamide gels were removed, maintaining alignment between the MWM and Eluate lanes. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007565#pone-0007565-g001" target="_blank"><i>Fig. 1B</i></a>) UV-visible spectra for 8 µM of as-isolated (black line) and reconstituted (red line) HydA1 apohydrogenase.</p