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[FeFe]-hydrogenases are efficient H 2 -catalysts, yet upon contact with dioxygen their catalytic cofactor (H-cluster) is irreversibly inactivated. Here, we combine X-ray crystallography, rational protein design, direct electrochemistry, and Fourier-transform infrared spectroscopy to describe a protein morphing mechanism that controls the reversible transition between the catalytic H ox -state and the inactive but oxygen-resistant H inact -state in [FeFe]-hydrogenase CbA5H of Clostridium beijerinckii . The X-ray structure of air-exposed CbA5H reveals that a conserved cysteine residue in the local environment of the active site (H-cluster) directly coordinates the substrate-binding site, providing a safety cap that prevents O 2 -binding and consequently, cofactor degradation. This protection mechanism depends on three non-conserved amino acids situated approximately 13 Å away from the H-cluster, demonstrating that the 1st coordination sphere chemistry of the H-cluster can be remote-controlled by distant residues. [FeFe]-hydrogenases catalyze the conversion of protons and electrons to molecular hydrogen, but upon exposure to oxygen, their catalytic cofactor is irreversibly inactivated. Here, the authors determine the crystal structure of hydrogenase CbA5H and identify a cysteine residue, which acts as a safety cap that shields the active site from oxygen.

Since their first industrial application in the acetone-butanol-ethanol (ABE) fermentation in the early 1900s, Clostridia have found large application in biomass biorefining. Overall, their fermentation products include organic acids (e.g., acetate, butyrate, lactate), short chain alcohols (e.g., ethanol, n-butanol, isobutanol), diols (e.g., 1,2-propanediol, 1,3-propanediol) and H 2 which have several applications such as fuels, building block chemicals, solvents, food and cosmetic additives. Advantageously, several clostridial strains are able to use cheap feedstocks such as lignocellulosic biomass, food waste, glycerol or C1-gases (CO 2 , CO) which confer them additional potential as key players for the development of processes less dependent from fossil fuels and with reduced greenhouse gas emissions. The present review aims to provide a survey of research progress aimed at developing Clostridium -mediated biomass fermentation processes, especially as regards strain improvement by metabolic engineering.

[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 O(2) 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.

Intradiol dioxygenase are iron-containing enzymes involved in the bacterial degradation of natural and xenobiotic aromatic compounds. The wild-type and mutants forms of catechol 1,2-dioxygenase Iso B from Acinetobacter radioresistens LMG S13 have been investigated in order to get an insight on the structure–function relationships within this system. 4K CW-EPR spectroscopy highlighted different oxygen binding properties of some mutants with respect to the wild-type enzyme, suggesting that a fine tuning of the substrate-binding determinants in the active site pocket may indirectly result in variations of the iron reactivity. A thermostability investigation by optical spectroscopy, that reports on the state of the metal center, showed that the structural stability is more influenced by the type rather than by the position of the mutation. Finally, the influence of pH and temperature on the catalytic activity was monitored and discussed in terms of perturbations induced on the tertiary contact network of the enzyme.

[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 O(2) 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.[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 O(2) 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.

Laboratory evolution techniques are becoming increasingly widespread among protein engineers for the development of novel and designed biocatalysts. The palette of different approaches ranges from complete randomized strategies to rational and structure-guided mutagenesis, with a wide variety of costs, impacts, drawbacks and relevance to biotechnology. A technique that convincingly compromises the extremes of fully randomized vs. rational mutagenesis, with a high benefit/cost ratio, is saturation mutagenesis. Here we will present and discuss this approach in its many facets, also tackling the issue of randomization, statistical evaluation of library completeness and throughput efficiency of screening methods. Successful recent applications covering different classes of enzymes will be presented referring to the literature and to research lines pursued in our group. The focus is put on saturation mutagenesis as a tool for designing novel biocatalysts specifically relevant to production of fine chemicals for improving bulk enzymes for industry and engineering technical enzymes involved in treatment of waste, detoxification and production of clean energy from renewable sources.

The technological innovation in the field of lighting and the need to reduce energy consumption connected to public lighting are leading many municipalities to undertake the renewal of public lighting systems, by replacing the existing luminaires with LED technologies. This renovation process is usually aimed at increasing energy efficiency and reducing maintenance costs, whist improving the lighting performance. To achieve these results, the new luminaires are often characterised by a luminous flux distribution much more downward oriented, which may remarkably influence and alter the perception of the night image of the sites. In this study the implications of the renovation of public lighting systems in terms of lighting and energy performance as well as the effects relating to the alteration of the night image, in historical contexts characterized by significant landscape value, are analysed. Results, along with demonstrating the positive effect that more sustainable and energy efficient lighting systems may have on the lighting performance and energy consumptions of public lighting systems, evidences the impact they may have on the alteration of the nocturnal image.