*3.3. [FeS]-Cluster Hydrogenases*

− − Hydrogenases are a class of [FeS] cluster-based metalloproteins which reversibly catalyze the two-electron reactions of dihydrogen oxidation and evolution [71,74]. The commonly reported membrane-bound hydrogenases can be classified based on their intrinsic catalytic cofactors ([FeFe], [NiFe], and [NiFeSe]), where hydrogen conversion is combined with [FeS] electron relay to accomplish the entire ET process. It has been reported that the smallest [FeFe]-hydrogenase *Cr*HydA1 isolated from *Chlamydomonas reinhardtii* exhibits dioxygen insensitivity and shows high catalytic activity in dihydrogen evolution [160]. The conditions of immobilized [FeFe]-hydrogenase *Cr*HydA1 on a SAM-modified gold electrode were characterized by in situ surface-enhanced infrared absorption spectroscopy (SEIRAS) and SPR spectroscopy [160]. Madden and associates investigated the catalytic activity of [FeFe]-hydrogenase CaHydA from *Clostridium acetobutylicum* immobilized on negatively charged MHA-modified Au(111) electrodes [74]. Electrochemical STM showed that the apparent height of [FeFe]-hydrogenase CaHydA continuously increased when the potential of the STM substrate was shifted from −0.4 to −0.6 V (vs. Ag/AgCl electrode), in which the hydrogen evolution response

was observed by CV. Notably, the catalysis of hydrogenase is easily quenched by carbon monoxide and cyanide binding to the Fe active sites.

Membrane-bound [NiFe]-hydrogenase and [NiFeSe]-hydrogenase have attracted considerable attention due to their resistance to dioxygen, carbon monoxide, as well as to high temperature [161]. Armstrong and associates reported a number of studies into the oxygen tolerance of [NiFe]-hydrogenase [162,163]. Typically, the catalytic sites of well-known hydrogenases are quenched by dissolved oxygen gas due to the high oxygen sensitivity of the internal peptide chains. Modification of Fe-S clusters by changing two oxygen-sensitive cysteine residues into glycines showed significant improvements in the long-term dihydrogen oxidation performance [162]. The authors demonstrated that the mechanism of oxygen tolerance mainly relates to removal of oxide species rather than preventing oxygen access into the protein. Lojou and associates demonstrated that carboxyl-terminated SAMs were favorable for optimizing the orientations of [NiFe]-hydrogenase from *Aquifex aeolicus* for efficient DET-type and MET-type bioelectrocatalysis of hydrogen oxidation, whereas hydrophobic SAMs only resulted in a MET process with the need for methylene blue mediator [71]. The electrochemical behavior of [NiFe]-hydrogenase from *Allochromatium vinosum*, *DesulfoVibrio Vulgaris* Miyazaki F (*Dv*MF), and *Ralstonia eutropha* H16 has also been reported [164–166].

Immobilization of membrane-bound hydrogenase on the SAM-modified electrode surface appears promising, but the ET progress of membrane-bound hydrogenase is challenging due to the complex enzyme structure compared with soluble redox enzymes. Gutiérrez-Sánchez and coworkers demonstrated that the introduction of a phospholipidic bilayer on positively charged 4-ATP modified Au electrode surfaces effectively controls the orientation of membrane-bound [NiFeSe]-hydrogenase for hydrogen oxidation (Figure 8) [167]. The hydrophobic lipid tail of hydrogenase can be embedded into the phospholipidic bilayer, thereby reducing the orientation distribution and promoting the ET process. All these results suggest that SAM-modified electrodes are paramount to provide a versatile platform for understanding how to tune the right enzyme orientation for DET.

**Figure 8.** Schematic illustration of [NiFeSe]-hydrogenase covalently inserted into a phospholipidic bilayer. Reproduced with permission from [167]. Copyright 2011, American Chemical Society.
