All biological processes require presence of specific enzymes. Processes of reduction of protons as well as oxidation of hydrogen (see reaction below)require at least presence of three enzymes: iron hydrogenase, nickel-iron hydrogenase and nitrogenase.

H2 ^ 2H+ + 2e — (1)

Hydrogenase exists in a number (~ 40) of prokariota, both aerobic and anaerobic, as well as in certain eukariota, e. g. in photosynthetic algae (Nickolet, 2000). Hydrogenases show different but significant sensitivity towards oxygen and light. Among more than 100 discovered hydrogenases essentially only those containing Fe and Ni atoms in active center are considered as the most attractive:

— [Fe] hydrogenase — containing only Fe atoms is the most sensitive towards oxygen inhibition but almost 100 times more active than [Ni-Fe] hydrogenases

— [Ni-Fe] or [Ni-Fe-Se] — these two types of hydrogenases indicate much higher affinity towards hydrogen than [Fe] hydrogenase (Darensbourg, 2000).

Active centers of both hydrogenases are composed from iron-sulfur clusters coordinated by carbonyl (CO) or cyanide (CN-) ligands.

Iron hydrogenases are the two directions enzymes because they catalyze both reduction of protons to the molecular hydrogen and the reverse reaction. There are three forms of these enzymes: monomeric — build only from the subunit controlling catalysis, dimeric, trimeric and tetrameric. Active centers located in these enzymes are not uniform either, however, all of them contain H-cluster (see Fig. 4) (Nicolet, 2000). Applying FTIR, EPR and XRD spectroscopy for analysis of monomeric hydrogenase, isolated from Clostridium pasterianum, it was found that H-cluster is composed from two basic units: [4Fe-4S] single group, responsible for electron transport, and the unique arrangement of [2Fe] capable to perform the reverse oxidation reaction of hydrogen. The regular cluster [4Fe-4S] is linked with four cysteine and sulfur atom of one of these forms the bridge bond between [4Fe-4S] and [2Fe]. In this dimeric system, the octahedral iron atoms are linked through two sulfur atoms (see Fig. 5) (Darensbourg, 2000). Moreover, it was found that these atoms are coordinated with five non-protein ligands (CO and CN-1) and water molecule. The bridge sulfur atoms forms additionally the 1,3- propanodithiol structure. The presence of covalent bond between sulfur atoms influence the charge of H-cluster and electric properties (Nicolet, 2000).

Fig. 4. Scheme of iron hydrogenase in Desulfovibrio desulfuricans (Dd) and Clostridium pasterianum (Cp). F — double cluster of [4Fe-4S], L-large subunit of H-cluster, S — small subunit of H cluster, Fd — [2Fe-2S] cluster related to ferredoxin. Pink color represents the unique structure of [4Fe-4S]. In Dd hydrogenase large and small subunits are connected via cysteine, whereas in Cp hydrogenase these units are linked with protein chain.

In active center of hydrogenase it is possible to identify such aminoacids as methionine and histidyne (Das, 2006). These two amino acids become attached to active center during formation of channels (for H2 and H+) connecting enzyme surface with reaction slit. The comparison of H-clusters in two strains of bacteria Clostridium pasteurianum (Cp) and Desulfovibrio desulfuricans (Dd) shows that in both cases the [2Fe] group is involved in hydrogen bond formation with lysine. However, when the second iron atom in Cp is engaged with serine, in the case of Dd, alanine is involved instead. In the case of fermentative bacteria of the Clostridium family in the large unit of monomeric iron hydrogenase it was confirmed a presence of three excessive systems: the [2Fe-2S] structure, rarely existing [4Fe-4S] structure with slit and space constructed from two [4Fe-4S] systems (Vignais, 2006).

Nickel-iron hydrogenase isolated from Desulfovibrio gigas and Desulfofibrio vulgaris is composed from large subunit a (60 kDa) containing Ni-Fe active center and small subunits в (30 kDa) equipped with three iron-sulfur clusters. These clusters are involved in electron transfer between active centers, donors and acceptors. All these clusters are located in the strait lines in which [3Fe-4S] appears between two [4Fe-4S] structures (Vignais, 2006).

The active center f [Ni-Fe] hydrogenase exhibits the unique location of ligands (see Fig. 6)

Here, four molecules of cysteine coordinate one three valent nickel atom. Two of them coordinate simultaneously iron, also located in active center. This kind of arrangement induce formation of sulfur bridges between nickel and iron atoms. Moreover, non-protein ligands such as SO, CO, CN and CO, CN are located in active centers of D. vulgaris and D. gigas, respectively. Nickel and iron atoms are bonded with monoatomic sulfur (D. vulgaris) or oxygen (D. gigas) bridges. Generated space is an ideal place for hydrogen reduction with electrons transported by iron-sulfur clusters from the surface of enzyme. The change of nickel valance form III to 0 and the return to basic state together with reconstruction of sulfur (or oxygen) bridge is observed in this catalytic cycle.

Nitrogenase is considered as the essential part of nitrogen circulation system in the living world. Nitrogen present in the air, needs to be transformed into compounds acceptable by living organisms. The diazotrophic microorganisms, including the PNS bacteria, are able to transform atmospheric nitrogen into NH3. There three types of nitrogenases built of two separate protein units: dinitrogenase (either Mo-Fe protein, or V-Fe protein, or Fe-Fe protein) and reductase (Fe protein). The main task of reductase is the delivery of electrons of high reductive potential to nitrogenase which uses them to different reduce N2 to NH3. Six electrons are involved in this process to reduce the oxidation degree of nitrogen from 0 to 3. The enzyme also transfers two other extra electrons to protons with final formation of one molecule of H2. Reduction of nitrogen to ammonia is highly energy consuming process because of the necessity of breaking the stable triple bond in nitrogen molecule and needs 16 ATP molecules per one molecule of nitrogen:

N2 + 8H+ + 8e + 16ATP ^ 2NH3 + H2 + 16ADP +16Pi (2)

Both components, nitrogenase and reductase are iron-sulphur proteins, in which iron is bonded with sulphur both in cysteine and the inorganic sulphide.

Reductase (Figure 7) is a dimer with mass of 30 kDa composed of four iron atoms and four inorganic sulphides (4Fe-4S). The site for ATP/ ADP bounding is located on the surface of this subunit. Reductase transfers electrons from the reduced ferredoxin towards dinitrogenase. This process occurs during hydrolysis of ATP with simultaneous dissociation of the complex.

Fig. 7. Reductase structure — nitrogenase component (Berg, 2002).

Dinitrogenase is a tetramer of the structure a2p2 and molecular weight of 240kDa (Figure 8). At the interface between the a and в subunits there is the P unit through which electrons are able to penetrate. Two cubo-octahedrons of 4Fe-4S are linked via sulphur atoms from cysteine residues. The flow electrons is realized from P unit to coenzyme Fe-Mo. This coenzyme is built of two units of M-3Fe-3S linked via sulphur atoms. In one unit M stands for Mo, while in the other one for Fe. Atmospheric nitrogen is transformed in the central part of coenzyme Fe-Mo. Multiple interactions of Fe-N type weaken the triple bond in molecular nitrogen which lowers the activation limit for nitrogen reduction (Berg, 2002). The synthesis of nitrogenase strongly depends on the light access to the medium and its intensity. Catalytic stability of nitrogenase is ensured by alternating light and dark 12-hour periods (day and night sequence) (Meyer, 1978). In the absence of molecular nitrogen and with large quantities of energy provided by ATP (Koku, 2002) nitrogenase catalyses hydrogen generation (see eq.3). Nitrogenase acts as a safety valve regulating cell reduction potential (Kars, 2010).

2H+ + 2e + 4ATP ^ H2 + 4ADP +4Pi (3)

There are two main inhibitors of nitrogenase during hydrogen photobiogeneration: molecular oxygen and nitrogen. In the presence of molecular nitrogen occurs competitive nitrogen fixation reaction and this stops almost completely hydrogen evolution. Ammonium ions at concentrations higher than 20 gmol are successful but reversible inhibitors of hydrogen generation (Waligorska, 2009) as well. The nitrogen necessary for the cell functioning is usually provided by ethanolamine and glutamate.

Fig. 8. Dinitrogenase construction (Berg, 2002).

However, glutamate can be the source of nitrogen inhibiting hydrogen evolution similarly as non-ammonium compounds. It can when glutamate becomes the source of carbon after the other sources are exhausted (Koku, 2002). In order to avoid such situation a medium with a relatively high ratio of organic carbon to nitrogen should be applied (e. g. malate to glutamate =15/2 (Eroglu, 1999).