*2.1. Membrane Enzymes in Biofuel Conversion*

Nature offers highly specialised enzyme machineries that can be exploited for (bio)fuel conversion. Among them, membrane-bound hydrogenases, found in many bacteria, archaea and lower eukaryotes, are metalloenzymes capable of catalysing the reversible oxidation of H<sup>2</sup> to protons and electrons [6]. Depending on the metal located in the active site, three phylogenetically unrelated classes can be identified: [NiFe], [FeFe] and, less common, [Fe] hydrogenases. Most hydrogenases are rapidly and almost completely inactivated by O<sup>2</sup> [7]. However, aerobic or facultative aerobic H2-oxidising bacteria have a particular subtype of O2-tolerant [NiFe] hydrogenase, which withstands the presence of O<sup>2</sup> and are membrane-bound hydrogenases (MBHs) [8]. The most studied O2-tolerant [NiFe] hydrogenases are found in *Ralstonia eutropha*, *Ralstonia metallidurans*, *Aquifex aeolicus*, *Hydrogenovibrio marinus* and *Escherichia coli*. MBHs are multimeric proteins with one large subunit and one small subunit, bound to the periplasmic side of the cytoplasmic membrane through a transmembrane protein (*b*-type cytochrome) [8]. The [NiFe] active site, with the same configuration as other [NiFe] hydrogenases, is deeply buried in the large subunit. MBHs couple oxidation of hydrogen in the large subunit to the reduction of either menaquinone-7 or ubiquinone-8 in the *b*-type cytochrome in the membrane.

In the respiratory chain, terminal oxidases catalyse the reduction of molecular oxygen to water without formation of reactive oxygen species (ROS). They oxidise quinones (ubiquinone and menaquinone oxidases) or cytochromes (cytochrome *c* oxidases) and can be classed into haem-copper oxidases [9], *bd* oxidases [10] or alternative oxidases [11]. For instance, cytochrome *bo*<sup>3</sup> enzymes are haem-copper oxidases in bacteria such as *E. coli* and couple oxidation of ubiquinol-8 to the reduction of oxygen [12]. Cytochrome *bd* is a quinol-dependent terminal oxidase found exclusively in prokaryotes and is structurally unrelated to haem-copper oxidases [13].

− − Nitrate reductases are molybdoenzymes capable of reducing nitrate (NO<sup>3</sup> <sup>−</sup>) to nitrite (NO<sup>2</sup> −). This enzyme family can be found in eukaryotic and prokaryotic cells. Eukaryotic nitrate reductases are present in plants, algae and fungi, and are involved in the assimilation of nitrate. Prokaryotic nitrate reductases are classified into three classes: assimilatory nitrate reductases (Nas), periplasmic nitrate reductases (Nap) and respiratory nitrate reductase (Nar). The latter are transmembrane enzymes which use nitrate as the electron acceptor of an anaerobic respiratory chain [14]. Facultative anaerobic bacteria use these alternative respiratory enzymes in oxygen depleted environments, replacing oxygen with different electron acceptors [15]. For instance, under anaerobic conditions, *E. coli* expresses a respiratory membrane-bound NarGHI coupled with a membrane-bound formate dehydrogenases (FDH-N). These two membrane enzymes form the supermolecular formate:nitrate oxidoreductase system. FDH-N oxidises formate to CO2, after which electrons are transferred to nitrate with the formation of a proton-motive force across the cytoplasmic membrane [16].
