There are two classes of hydrogenases that commonly present in phototrophic organisms: the [Fe—Fe]-

hydrogenase and the [Ni—Fe]-hydrogenase. The [Fe—Fe]- hydrogenase is found in green algae and some bacteria, and it is the most active H2-forming enzyme. It demon­strates about 100 times higher activity than the [Ni—Fe]- hydrogenase. However, it is irreversibly inactivated when exposed to O2 (see Section Green Algal [Fe—Fe]-Hy — drogenases in this chapter). Cyanobacteria possess two types of [Ni—Fe]-hydrogenases (Houchins, 1984), which are more tolerant to O2 and only temporarily inactivated upon exposure to O2.

Uptake Hydrogenases

In cyanobacteria, the uptake hydrogenases (encoded by hupSL genes) catalyze the consumption of H2 pro­duced by the nitrogenase. Thus, the net H2 evolution by N2-fixing cyanobacteria is barely observed under nat­ural conditions. Uptake hydrogenase has been found in all N2-fixing cyanobacteria studied so far. Nevertheless, a few N2-fixing Synechococcus strains lacking an uptake hydrogenase have been reported (Ludwig et al., 2006; Steunou et al., 2008). It is believed that the uptake hydrogenase transfers electrons from H2 back to the photosynthetic and respiratory electron transport chains, and thus partially regains the energy used for N2 fixation. The cellular/subcellular localization of the uptake hydrogenase is controversial and seems to be species specific. The data obtained from the N2-fixing filamentous nonheterocystous cyanobaterium, Lyngbya majuscule, revealed higher specific labeling associated with the thylakoid membranes, suggesting that the cya — nobacterial uptake hydrogenase is a membrane-bound protein (Seabra et al., 2009). However, it lacks a membrane-spanning region. Therefore, the presence of the third subunit, which would anchor the uptake hy — drogenase to the membrane and link electron transfer from the enzyme to the respiratory or photosynthetic chains, has been suggested (Tamagnini et al., 2007).

In some heterocystous cyanobacteria, such as Anabaena PCC 7120, the uptake hydrogenase enzyme was detected only in heterocysts, while in other cyano­bacteria, such as Nostoc punctiforme, it is localized in both vegetative and heterocyst cells, corresponding most probably to inactive and active pools of the enzyme (Camsund et al., 2011; Seabra et al., 2009).

Since the uptake hydrogenase is an obstacle for H2 production, mutations disrupting the structural hupSL genes have been constructed to improve the H2 produc­tion in N2-fixing cyanobacteria (Happe et al., 2000; Lindberg et al., 2002; Masukawa et al., 2002; Schutz et al., 2004; Yoshino et al., 2007; Khetkorn et al., 2012). These mutants produced about four — to sevenfold more H2 than the control strain. In addition, inactivation of uptake hydrogenase had no major effect on cell growth and heterocyst differentiation. Quantitative shotgun proteomics and physiological approaches on the uptake hydrogenase mutant of N. punctiforme demonstrated that the mutant strain undergoes meta­bolic and structural alterations to compensate for the amount of electrons lost as a release of H2 (Ekman et al., 2011). Construction of mutant strains combining several improvements is likely to be a better approach toward sustainable H2 production. To this end, the single — and the double-mutant strains lacking the homocitrate synthase genes, nifVl and nifV2, were con­structed using the DhupL strain of Anabaena PCC 7120 as the parental strain (Masukawa et al., 2007). The catalytic Mo—Fe center binds homocitrate, which is necessary for N2 fixation, but in the absence of homocitrate gene Mo—Fe center binds citrate: in a Klebsiella mutant this was shown to demonstrate low N2 fixation but high H2 production activity in a N2 atmosphere (Mayer et al., 2002). In line with this result, the DhupLDnifV1 cells also demonstrated high H2 production rate and heterocyst frequency compared to the parental DhupL in N2 atmosphere (Masukawa et al., 2007).