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[FeFe] hydrogenases rare Nature’s best H₂-processing catalysts, and one of the best candidates to satisfy societal need for cheap and efficient catalyst for H₂-evolution. These enzymes owe their remarkable catalytic activities to their organometallic active site, called “H-cluster”. The H-cluster can be described as a canonical [4Fe4S] cluster linked via a bridging cysteine residue to a [2Fe] subsite, which is in turn coordinated by three CO, two CN⁻ and one bidentate azadithiolate ligand. This unique cofactor allows these enzymes to function with virtually no overpotential requirements and TOFs up to 20 000 s^-1. We have now reached a good understanding of the catalytic cycle by which these enzymes operate, yet many questions remain open especially regarding the physiological relevance of some the proposed intermediates. 

In this thesis, we have used FTIR and EPR spectroscopies on whole-cell samples of [FeFe] hydrogenases to study the influence of the intracellular environment on the catalytic cycle of these enzymes. Moreover, we have investigated how the bacterial cytoplasm influences the stability of the H-cluster and favours the formation of sulfide-inhibited states, and we have studied the role of the proton-transfer chain and of steric factor related to the active site pocket in promoting this inhibition. Today, whole-cell systems attract a lot of interest due to the possibility to couple living cells with artificial photosensitizer to create whole-cell photocatalytic systems endowed with self-healing capabilities and broadband light absorption properties. We have built a system consisting of an E. coli-encapsulated [FeFe] hydrogenase coupled to the organic photosensitizer eosin Y, and we have verified the occurrence of light-induced electron transfer from eosin Y to the enzyme with consequent H₂ production. We have also applied a simple design-of-experiments approach to look into how the different system parameters interact with each other, and we have demonstrated that such approaches represent a viable strategy to guide the optimization of this type of photocatalytic systems. Finally, we have moved our attention to semi-artificial hydrogenases. We have used artificial maturation to generate asymmetric monocyanide versions of the H-cluster using two different model hydrogenases, CrHydA1 and DdHydAB. Through the application of FTIR, EPR and Protein-Film Electrochemistry we characterized these variants, studied the effect of this ligand exchange on the overall catalytic performance, and shed light on the effect of second-coordination sphere interaction on parameters such as catalytic rate, overpotential, affinity for H₂ and inhibitor sensitivity.