Hydrogen photoproduction in green algae is catalyzed by [Fe—Fe]-hydrogenases. Earlier reports have suggested the existence of [Ni—Fe]-hydrogenase in S. obliquus (Zinn et al., 1994), but presently S. obliquus is considered as having only the [Fe—Fe]-hydrogenase (Wunschiers et al., 2001; Florin et al., 2001). Some green algal species do not show hydrogenase activity at all (Brand et al., 1989; Boichenko and Hoffmann, 1994). Currently, the presence of genes encoding [Fe—Fe]-hydrogenases has been proved in the following species: Chlamydomonas reinhard — tii (Happe and Kaminski, 2002; Forestier et al., 2003), Chlorellafusca (Winkler et al., 2002), Chlamydomonas nocti — gama (Skjanes et al., 2010), Volvox carteri (Prochnik et al., 2010), Tetraselmis subcordiformis (Yan et al., 2011) and Chlorella variabilis (Meuser et al., 2011). Algal [Fe—Fe]- hydrogenases in vivo interact with Fd, a terminal acceptor of photosynthetic electron transport chain (Chang et al.,

2007) . In contrast to [Ni—Fe]-hydrogenase enzymes, [Fe—Fe]-hydrogenases have significantly higher turnover rate (6000—9000/s) and usually catalyze H2 production instead of H2 uptake (Frey, 2002). However, the possible role of these enzymes in H2 uptake under high H2 partial

21. RECENT DEVELOPMENTS ON CYANOBACTERIA AND GREEN ALGAE FOR BIOHYDROGEN PHOTOPRODUCTION

pressure has also been suggested (Kosourov et al., 2012). Unfortunately, the [Fe—Fe]-hydrogenases are extremely sensitive to O2 that irreversibly inactivates purified en­zymes within seconds (Ghirardi et al., 1997).

The most studied green alga, C. reinhardtii has two monomeric [Fe—Fe]-hydrogenases: HydA1 and HydA2 with a molecular mass of around 48 kD (Happe and Kaminski, 2002; Forestier et al., 2003). Both proteins are nuclear encoded and contain putative transit se­quences that target them to the chloroplast. The hydAl gene shows 74% similarity to hydA2 and encodes protein that is 68% identical to HydA2. Two homologous hydrogenases are typically observed in almost all green algae showing hydrogenase activity (Winkler et al., 2004). Nevertheless, some species have three [Fe—Fe]- hydrogenase enzymes (Skjanes et al., 2010). The phy­siological basis for the presence of two and more hydrogenases in green algae has not been determined. In C. reinhardtii cells, HydA1 most probably participates in the light-dependent H2 production pathway (Happe and Naber, 1993). Examination of relative enzyme activ­ities by gene-silencing techniques indicate that HydA1 catalyzes the majority of the hydrogenase activity, but the role of HydA2 in algal H2 production has not been clearly resolved (Godman et al., 2010). Recently, Meuser and et al. (2012) using the single hydAl, hydA2 and double hydA1/hydA2 knockout mutants showed that HydA2 also participates in H2 photoproduction. However, according to the authors, its contribution in the light-dependent process does not exceed 25%. The next important step in this direction should be investiga­tion of the role of these enzymes in the H2 uptake, including the mechanisms of photoreduction (Kosourov et al., 2012).