Hydrogenases in cyanobacteria have been studied for over 35 years (Benemann and Weare, 1974; Hallenbeck and Benemann, 1978) and many variations of hydroge — nases have been described in different bacterial phyla (Vignais and Billoud, 2007). These enzymes are frequently classified into three different groups: nitroge — nase, the reversible hydrogenase (Hox), and the uptake hydrogenase (Hup) (Ghirardi et al., 2007).

HUP—HYDROGEN UPTAKE ENZYME

Hup is a [NiFe] hydrogenase that occurs associated with the thylakoid membrane (Seabra et al., 2009). This enzyme shows the least sensitivity to oxygen among the three classes. Its function is in the oxidation of H2, returning the captured electrons to cellular electron transfer reactions. To date it has been found only in N2-fixing strains and appears to have an intimate rela­tionship with nitrogenase (Marreiros et al., 2013). Under natural conditions, nitrogenase functions to reduce atmo­spheric N2 to NH3, producing H2 in an unavoidable side reaction. It is thought that Hup functions to recycle the recently formed H2, which is oxidized back into protons or reacted with O2 in a respiratory oxyhydrogen reaction, protecting the nitrogenase from O2 inactivation, avoiding an excessive build up of H2 in the cell and recovering part of the ATP used in its formation (Bothe et al., 2010; Tamagnini et al., 2007). In the nitrogen-fixing cyanobacte­ria, transcription of the Hup-encoding genes hupSL is associated with the nitrogen depletion response and is under the regulation of the NtcA, the global nitrogen regulator (Weyman et al., 2008). Hup inactivation increases the production of H2 two — to threefold in most cyanobacteria (Ludwig et al., 2006; Tamagnini et al., 2007).

NITROGENASE—A GRATUITOUS HYDROGENASE

In nature this complex enzyme carries out a critical function, breaking the three covalent bonds of molecu­lar nitrogen (N2) providing ammonia to the cell and closing the nitrogen cycle. This process consumes a large amount of energy in the form of ATP and high — energy electrons (Eqn (22.1)), producing NH3 with the coproduction of hydrogen in an unavoidable side reaction.

N2 + 10H+ + 8e~ + 16ATP/2NH3 + H2 + 16ADP

(22.1)

The most common nitrogenase is the Mo-Fe nitrogenase, which is characterized by a complex iron-sulfur cluster containing molybdenum. While performing nitrogen fixation, up to one-fourth of the electron flux goes to­ward the reduction of hydrogen. Variations of this enzyme includes the substitution of the molybdenum by vanadium or iron (V-Fe and Fe—Fe nitrogenases, respectively), which, although a greater proportion of electrons are allocated to hydrogen production, in fact show a lower net flux of electrons to hydrogen since their overall reaction rates are much lower than that of the Mo-Fe enzyme, limiting the application of these variants in bioproduction systems. One option that is an interesting strategy for H2 production, to increase the electron flux into H2, is cultivation in the absence of N2, since nitrogenase turnover continues, but now the elec­tron flux goes totally toward hydrogen evolution. In addition, the growth arrest caused by the nutrient lim­itation is of interest as this decouples hydrogen evolu­tion from biomass production, therefore potentially leaving more energy available for H2 production (Benemann and Weare, 1974). Even so, the expression of an oxygen-sensitive enzyme in an O2 rich milieu is counter productive. To overcome this problem, temporal separation between N2 fixation and photosynthesis can be used, where during the day the photosynthetic machinery works toward the carbon fixation, which then can be consumed to power nitrogenase and consequently proton reduction. Interestingly, the peak of hydrogen production in indirect biophotolysis occurs when the cell is reilluminated, possibly due to a burst in ATP synthesis before the oxygen formed by PSII (Figure 22.1) reaches a toxic level for the nitrogenase.

Heterocyst forming species on the other hand can perform direct biophotolysis by carrying out nitrogen fixation in the differentiated cell during the day. The heterocyst can maintain an internal anoxic environment since the expression of PSII is repressed. Hydrogen production therefore is supported through the use of carbon compounds delivered by the neighboring vegetative cells.

REVERSIBLE HYDROGENASE (HOX)

In addition to nitrogenase, N2-fixing cyanobacteria can have a second hydrogen-evolving enzyme, the so-called reversible hydrogenase (Hox). This enzyme is a heteropentameric complex that is formed by a hydrog — enase module (HoxHY) and a diaphorase module (Hox — EFU), which transfers electrons from NAD(P)H to the hydrogenase module (Bothe et al., 2010). Like Hup, Hox is a [NiFe] hydrogenase, but in this case it shows a high sensitivity to O2. Its expression is totally indepen­dent from that of nitrogenase and varies among species. In some cases it is under the control of the circadian clock, where it is shown to promote hydrogen produc­tion in the dark (Hallenbeck and Benemann, 1978; Schmitz et al., 2001). The bidirectional hydrogenase is not taxon specific, being found in many different groups of cyanobacteria, and its location and organization in the chromosome are also heterogeneous. Recent studies regarding Hox transcription factors have elucidated many aspects of its regulatory mechanisms, which are reviewed elsewhere (Oliveira and Lindblad, 2009).