[FeFe]-hydrogenases are efficient H₂-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 O₂-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 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.

Catechol 1,2-dioxygenases are iron containing enzymes able to convert catechol into cis,cis-muconate, a precursor of the industrially important compound adipic acid. Catechol 1,2-dioxygenase from Acinetobacter radioresistens S13 was immobilized on β-cyclodextrins cross-linked with carbonate groups (nanosponges) with a yield of 29 mg of enzyme per gram of support. This support was chosen for its low cost and its ability to offer different types of interactions with the enzyme. The activity profiles at different pH and temperatures showed a shift of the optimal pH from 8.5, for the free protein, to 9.5, for the immobilized protein and, similarly, a shift in optimal temperature from 30 °C to 50 °C. The Michaelis–Menten constant, K_M, increased from 2.0 ± 0.3 μM, for the free form, to 16.6 ± 4.8 μM for the immobilized enzyme, whereas the rate constant, k_cat, values were found to be 32 ± 2 s⁻¹ and 27 ± 3 s⁻¹ for the free and immobilized forms respectively. The immobilization process also increased the thermostability of the enzyme with 60% residual activity after 90 min at 40 °C for the immobilized proteinversus 20% for the free enzyme. A residual activity of 75% was found after 15 min at 60 °C for the immobilized enzyme while the free form showed a total loss of activity under the same conditions. The activity toward other substrates, such as 3- and 4-methylcatechol and 4-chlorocatechol, was retained by the immobilized enzyme. A small scale bioreactor was constructed and was able to convert catechol into cis,cis-muconic acid with high efficiency for 70 days.

[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 TiO₂ electrode. The enzyme is able to interact and exchange electrons with the electrode and to catalyze hydrogen production with an efficiency of 70%.