Simone Morra

Site Saturation Mutagenesis Demonstrates a Central Role for Cysteine 298 as Proton Donor to the Catalytic Site in CaHydA (FeFe)-Hydrogenase

[FeFe]-hydrogenases reversibly catalyse molecular hydrogen evolution by reduction of two protons. Proton supply to the catalytic site (H-cluster) is essential for enzymatic activity. Cysteine 298 is a highly conserved residue in all [FeFe]-hydrogenases; moreover C298 is structurally very close to the H-cluster and it is important for hydrogenase activity. Here, the function of C298 in catalysis was investigated in detail by means of site saturation mutagenesis, simultaneously studying the effect of C298 replacement with all other 19 amino acids and selecting for mutants with high retained activity. We demonstrated that efficient enzymatic turnover was maintained only when C298 was replaced by aspartic acid, despite the structural diversity between the two residues. Purified CaHydA C298D does not show any significant structural difference in terms of secondary structure and iron incorporation, demonstrating that the mutation does not affect the overall protein fold. C298D retains the hydrogen evolution activity with a decrease of k cat only by 2-fold at pH 8.0 and it caused a shift of the optimum pH from 8.0 to 7.0. Moreover, the oxygen inactivation rate was not affected demonstrating that the mutation does not influence O2 diffusion to the active site or its reactivity with the H-cluster. Our results clearly demonstrate that, in order to maintain the catalytic efficiency and the high turnover number typical of [FeFe] hydrogenases, the highly conserved C298 can be replaced only by another ionisable residue with similar steric hindrance, giving evidence of its involvement in the catalytic function of [FeFe]-hydrogenases in agreement with an essential role in proton transfer to the active site.

Direct electrochemistry of an [FeFe]-hydrogenase on a TiO2 electrode.

[FeFe]-hydrogenases are efficient natural catalysts that can be exploited for hydrogen production. Immobilization of the recombinant [FeFe]-hydrogenase CaHydA was achieved for the first time on an anatase TiO(2) electrode. The enzyme is able to interact and exchange electrons with the electrode and to catalyze hydrogen production with an efficiency of 70%.

The effect of a C298D mutation in CaHydA [FeFe]-hydrogenase: Insights into the protein-metal cluster interaction by EPR and FTIR spectroscopic investigation.

A conserved cysteine located in the signature motif of the catalytic center (H-cluster) of [FeFe]-hydrogenases functions in proton transfer. This residue corresponds to C298 in Clostridium acetobutylicum CaHydA. Despite the chemical and structural difference, the mutant C298D retains fast catalytic activity, while replacement with any other amino acid causes significant activity loss. Given the proximity of C298 to the H-cluster, the effect of the C298D mutation on the catalytic center was studied by continuous wave (CW) and pulse electron paramagnetic resonance (EPR) and by Fourier transform infrared (FTIR) spectroscopies. Comparison of the C298D mutant with the wild type CaHydA by CW and pulse EPR showed that the electronic structure of the center is not altered. FTIR spectroscopy confirmed that absorption peak values observed in the mutant are virtually identical to those observed in the wild type, indicating that the H-cluster is not generally affected by the mutation. Significant differences were observed only in the inhibited state Hox-CO: the vibrational modes assigned to the COexo and Fed-CO in this state are shifted to lower values in C298D, suggesting different interaction of these ligands with the protein moiety when C298 is changed to D298. More relevant to the catalytic cycle, the redox equilibrium between the Hox and Hred states is modified by the mutation, causing a prevalence of the oxidized state. This work highlights how the interactions between the protein environment and the H-cluster, a dynamic closely interconnected system, can be engineered and studied in the perspective of designing bio-inspired catalysts and mimics.

Hydrogen production at high Faradaic efficiency by a bio-electrode based on TiO2 adsorption of a new [FeFe]-hydrogenase from Clostridium perfringens

The [FeFe]-hydrogenase CpHydA from Clostridium perfringens was immobilized by adsorption on anatase TiO2 electrodes for clean hydrogen production. The immobilized enzyme proved to perform direct electron transfer to and from the electrode surface and catalyses both H2 oxidation (H2 uptake) and H2 production (H2 evolution) with a current density for H2 evolution of about 2 mA cm(-1). The TiO2/CpHydA bioelectrode remained active for several days upon storage and when a reducing potential was set, H2 evolution occurred with a mean Faradaic efficiency of 98%. The high turnover frequency of H2 production and the tight coupling of electron transfer, resulting in a Faradaic efficiency close to 100%, support the exploitation of the novel TiO2/CpHydA stationary electrode as a powerful device for H2 production.

Oxygen Stability in the New [FeFe]-Hydrogenase from Clostridium beijerinckii SM10 (CbA5H)

The newly isolated Clostridium beijerinckii [FeFe]-hydrogenase CbA5H was characterized by Fourier transform infrared spectroscopy coupled to enzymatic activity assays. This showed for the first time that in this enzyme the oxygen-sensitive active state Hox can be simply and reversibly converted to the oxygen-stable inactive Hinact state. This suggests that oxygen sensitivity is not an intrinsic feature of the catalytic center of [FeFe]-hydrogenases (H-cluster), opening new challenging perspectives on the oxygen sensitivity mechanism as well as new possibilities for exploitation in industrial applications.

Isolation and characterization of a new [FeFe]‐hydrogenase from Clostridium perfringens

This paper reports the first characterization of an [FeFe]‐hydrogenase from a Clostridium perfringens strain previously isolated in our laboratory from a pilot‐scale bio‐hydrogen plant that efficiently produces H2 from waste biomasses. On the basis of sequence analysis, the enzyme is a monomer formed by four domains hosting various iron–sulfur centres involved in electron transfer and the catalytic center H‐cluster. After recombinant expression in Escherichia coli, the purified protein catalyzes H2 evolution at high rate of 1645 ± 16 s−1. The optimal conditions for catalysis are in the pH range 6.5–8.0 and at the temperature of 50 °C. EPR spectroscopy showed that the H‐cluster of the oxidized enzyme displays a spectrum coherent with the Hox state, whereas the CO‐inhibited enzyme has a spectrum coherent with the Hox‐CO state. FTIR spectroscopy showed that the purified enzyme is composed of a mixture of redox states, with a prevalence of the Hox; upon reduction with H2, vibrational modes assigned to the Hred state were more abundant, whereas binding of exogenous CO resulted in a spectrum assigned to the Hox‐CO state. The spectroscopic features observed are similar to those of the [FeFe]‐hydrogenases class, but relevant differences were observed given the different protein environment hosting the H‐cluster.

Atypical effect of temperature tuning on the insertion of the catalytic iron-sulfur center in a recombinant [FeFe]-hydrogenase.

The expression of recombinant [FeFe]‐hydrogenases is an important step for the production of large amount of these enzymes for their exploitation in biotechnology and for the characterization of the protein‐metal cofactor interactions. The correct assembly of the organometallic catalytic site, named H‐cluster, requires a dedicated set of maturases that must be coexpressed in the microbial hosts or used for in vitro assembly of the active enzymes. In this work, the effect of the post‐induction temperature on the recombinant expression of CaHydA [FeFe]‐hydrogenase in E. coli is investigated. The results show a peculiar behavior: the enzyme expression is maximum at lower temperatures (20°C), while the specific activity of the purified CaHydA is higher at higher temperature (30°C), as a consequence of improved protein folding and active site incorporation.

EXTRACTION OF BIOCHEMICALS FROM THE WINE INDUSTRY BY-PRODUCTS AND THEIR VALORIZATION

The production process of wine and distilled generates several by-products (20-30% of total production). Currently, most of the solid by-products (pomace, stalks, lees and seeds) obtained as downstream of the vinery industry, are conferred to the distillery or, less frequently, used in agriculture and for energy production. As a consequence, much of the antioxidant compounds contained in the grape is unused in the products of processing and is lost. These substances (among which the most important are polyphenols, anthocyanins and resveratrol) are a heterogeneous group of compounds particularly known for their beneficial effects on human health. In this article we present the results arising from a pilot scale research devoted to the evaluation of the extraction of such important compounds from the by-products of four varieties of Italian grape varieties. The pomaces obtained after wine production were extracted by innovative technologies, such as steam explosion and enzymatic extraction, without the use of organic solvents. The results show that it is possible to recover relevant amounts of polyphenols (up to 1383±50 mg GAE/L), anthocyanins (up to 148±2 mg/L) and resveratrol (up to 0.064 mg/L) from such by-products. Moreover, the recovered biochemicals are functional and act as radical scavenger, suggesting possible future applications in the cosmetics industry. The novel approach proposed here supports the possible application of steam explosion as industrial techniques to recover valuable compounds from grape pomace in a sustainable perspective from the economic and environmental standpoint.