The bidirectional hydrogenases can either produce or consume H2 according to the cellular redox environ­ment. The enzyme functions in dark fermentation and under specific conditions in photoproduction of H2.

In cyanobacteria the bidirectional hydrogenase con­sists of two structural moieties: the hydrogenase (encoded by hoxYH) and the diaphorase unit (encoded by hoxUFE) capable of oxidation of NAD(P)H. Over the last several years, significant progress has been achieved in the identification of the transcription factors, such as LexA and AbrB-like proteins, which are members of the complex signal cascade that directs the expression of the bidirectional hydrogenase genes (Oliveira and Lindblad, 2009).

On the basis of high sequence similarity, it has been hy­pothesized that the diaphorase subunit of the bidirec­tional hydrogenase also serves as the three missing activity subunits of cyanobacterial respiratory NDH-1 complex (Appel and Schulz, 1996). However, more recent results have not supported this hypothesis since the mu­tants lacking the diaphorase subunits do not show mal­function of the respiratory activity (Boison et al., 1999).

Bidirectional hydrogenase has been found in all non-N2-fixing and some N2-fixing cyanobacteria. Thus, many filamentous N2-fixing cyanobacteria contain both the bidirectional and the uptake hydrogenase. However, a few species have only the uptake hydrogenase. In cyanobacteria, the bidirectional hydrogenase is constitu­tively expressed under both aerobic and anaerobic condi­tions but is active only in the dark, anoxic conditions or during the transition from dark to light (Cournac et al., 2004; Schutz et al., 2004). The biological function of bidi­rectional hydrogenase in filamentous cyanobacteria is not well understood (Tamagnini et al., 2007). Mutational studies with hox-defective mutants suggested that the bidirectional hydrogenase in N2-fixing cyanobacteria does not support N2 fixation (Masukawa et al., 2002). In non-N2-fixing cyanobacteria the bidirectional hydroge — nase is the main H2-producing enzyme and it is thought to interact with photosynthetic pathways (Ludwig et al.,

2006) . However, H2 production catalyzed by bidirectional hydrogenases is only transient (less than 30 s in light) since it is quickly inhibited by increasing photosynthetic O2 evolution (Cournac et al., 2004). In line with this, no transient H2 evolution was detected in different Hox deletion mutants studied by Aubert-Jousset et al. (2011). It is hypothesized that the bidirectional hydrogenase functions as a safety electron sink thereby removing excess reducing equivalents during the dark, anaerobic to light transition in unicellular Synechocystis cells (Appel et al., 2000; McIntosh et al., 2011). This hypothesis is inter­esting due to the natural environment of cyanobacteria being highly dynamic, with rapid fluctuations in light in­tensity. Such fluctuations might strongly unbalance the function of the photosynthetic complexes, resulting in production of reactive oxygen species and destroying photosynthetic apparatus. Cyanobacteria have unique flavodiiron proteins, Flv1 and Flv3, functioning as a strong electron sink at the end of light reactions by direct­ing excess electrons to O2 without production of reactive oxygen species, thus maintaining the redox balance of the electron transport chain (Helman et al., 2003; Allahver — diyeva et al., 2011, 2012). A bidirectional hydrogenase possibly takes over the role of a strong electron sink upon dark to light transitions during anaerobiosis, the condition created in cyanobacterial mats and blooms, and where the Flv1 and Flv3 pathway is not functional (Gutthann et al., 2007).

In Synechocystis, both NADPH and NADH can act as electron donors for the bidirectional hydrogenase. Recent studies showed that NADH is a preferential sub­strate of the diaphorase moiety, whereas NADPH is an efficient activator of the bidirectional hydrogenase (Aubert-Jousset et al., 2011). These results are in line with the observed dynamics of H2 production during dark—light transition. In the dark anaerobic conditions, H2 is produced by oxidation of NADH, the major prod­uct of glycolysis assimilation. Sudden exposure to light produces NADPH by photosynthetic electron transfer chain, which functions as an activator of the hydroge — nase and begins consumption of H2. Although there is no strong evidence for direct electron donation from reduced Fd (E0 = —0.42 V), which is a stronger reductant than NADH (E0 = —0.315 V), to the bidirectional hydrogenase, such an electron transfer cannot be completely excluded (McNeely et al., 2011). Direct linkage of Fd to the bidirectional enzyme in mutants lacking the diaphorase domain could be employed to improve cyanobacterial H2 production.

Accounting for the high affinity of the bidirectional hydrogenase to H2, it has been suggested that the enzyme can function in utilization of H2 under physio­logical conditions. However, it should be kept in mind that the bidirectional hydrogenase reversibly evolves H2 under dark, anaerobic conditions as a result of fermentation of photosynthetically stored carbon inter­mediates in cyanobacteria. Recent electrochemical investigations of the bidirectional enzyme from Synecho­cystis PCC 6803 have revealed unexpected properties. The rate of H2 production at low pH and low H2 pres­sure was shown to be about 1.4 times faster than the rate of H2 consumption at high pH and high H2 pressure (McIntosh et al., 2011).