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

Comparing this work to previous reports for the production of the <i>C. reinhardtii</i> HydA1 hydrogenase.

1<p>Specific activities for H₂ production rates are expressed in units of µmol H₂·min⁻¹·mg⁻¹ of HydA1.</p>2<p>Purification yields are mg of HydA1 isolated per liter of cell culture.</p>3<p>Iron content is expressed in units of Fe atoms per HydA1 peptide; NR, not reported.</p

Effects of iron and cysteine supplementation as well as the <i>iscR</i> deletion on active hydrogenase expression.

<p>Iron (2 mM ferric ammonium citrate) and cysteine (2 mM) were added to cultures as indicated. Methyl viologen reducing activities (µmol MV reduced·min⁻¹·mg⁻¹ total protein) of active [FeFe] hydrogenase in cell lysates from <i>E. coli</i> strains BL21(DE3) (gray bars) and BL21(DE3) <i>ΔiscR</i> (black bars). Hydrogenase activities were measured after 16–18 hrs of anaerobic HydA1 expression (A) and CpI expression (B). Hydrogenase activities for the <i>ΔiscR</i>-derived samples are indicated above the respective columns, and all activities are the average for n = 2 cultures ± standard deviations.</p

Biophysical characterization of purified [FeFe] hydrogenases produced in <i>Escherichia coli</i>.

<p>Data are the average from n = 3–6 cultures examined ± standard deviations.</p>†<p>Specific activities of purified hydrogenases were determined using methyl viologen (MV) or PetF ferredoxin as the electron donating or accepting substrates.</p