Many cyanobacteria are able to fix atmospheric N2 into ammonia (NH3) and produce H2 as a by-product. The reaction of nitrogen fixation is catalyzed by nitroge — nase, a complex metalloenzyme and results in the formation of 1 mol of H2 per 1 mol of fixed N2 (Phelps and Wilson, 1942):

N2 + 8H+ + 8e~ + 16ATP / 2NH3 2 , 3 (21.2) + 16(ADP + PO+H2,

where Pi is inorganic phosphate.

Nitrogenases have relatively low turnover numbers. N2 fixation is an energy-expensive process that requires two ATP molecules per electron transfer. It has, howev­er, an advantage of catalyzing an irreversible reaction and not being inhibited by H2 accumulation.

The best studied type of nitrogenase is conventional molybdenum (Mo)-nitrogenase, which is encoded by the structural genes nifHDKl. Very little is known about the regulation of nif genes. Nitrogenase consists of two components: dinitrogenase, or the MoFe protein

composed of NifD and NifK subunits, and dinitrogenase reductase, or the Fe protein, consisting of two subunits of NifH. The substrate-binding and reducing active site is located in the MoFe protein. The Fe protein con­taining a [4Fe—4S] cluster and a Mg-ATP binding site acts as electron donor to a MoFe protein. This cluster accepts electrons from Fd or flavodoxin. The fixation of N2 is always accompanied by H2 evolution (Hadfield and Bulen, 1969). The reason for production of H2 as a by-product is not yet clear. It could be a result of unavoidable leakage of reducing potential, or formation of H2 could be a prerequisite for binding of N2 to the active site (Burgess and Lowe, 1996). Besides reducing N2, nitrogenase can reduce a number of other substrates with triple bonds. Importantly, in the absence of N2 as a substrate, nitrogenase exclusively catalyzes ATP-dependent reduction of H+ to H2 (Benemann and Weare, 1974; Pickett, 1996).

8H+ + 8e~ + 16ATP / 16(ADP + P;) + 4H2 (21.3)

Indeed, in terms of H2 production by N2 fixation in het­erocystous cyanobacteria the N2 is a much more potent inhibitor than O2 (Yeager et al., 2011). This is logical,
due to the fact that heterocysts can protect enzymes from external O2. Since the replacement of N2 with argon (Ar) gas is an expensive approach for optimi­zation of H2 production, an alternative method to genet­ically modify the catalytic site of the nitrogenase enzyme has been chosen as more appropriate. Recently, site — directed mutations have been introduced to several amino acid residues coordinating the Mo—Fe active site of the nzfl-enzyme in attempts to direct the electron flow selectively to H2 production in atmospheric N2 condition (Masukawa et al., 2010). Importantly, several mutant strains demonstrated nearly similar rate of H2 produc­tion under N2 and Ar atmosphere. Moreover, these strains accumulated significantly high levels of H2 under atmospheric N2 as compared to the reference strains.

Alternative Nitrogenases

In addition to the conventional Mo-nitrogenase, nifl, N2-fixing microorganisms possess also alternative nitro- genases: second type of Mo-nitrogenase, nif2, vanadium (V)-nitrogenase, vnf, and Fe-nitrogenase, anf (Bothe et al., 2010). Presence of the Fe-nitrogenase in cyanobac­teria has not yet been documented.

Mo-nitrogenase 2, encoded by nifHDK2, is expressed in both vegetative cells and heterocysts of Anabaena variabilis under N2-fixing and anaerobic conditions (Schrautemeier et al., 1995; Thiel et al., 1995). Unicellular Chroococcidiopsis, inhabiting in a gypsum rock, where the shards provide a microaerobic, low light environment, also possesses the alternative nif 2 system. Based on the phylogenetic analysis of nifH sequences, it has been suggested that nif 2 is characteristic of unicellular or fila­mentous nonheterocystous cyanobacteria fixing N2 only under microaerobic conditions (Boison et al., 2004). Weyman and coworkers reported the amino acid substi­tution in nifD2 as a first step toward the development of nitrogenase mutants in A. variabilis, which produces large amounts of H2 in N2 containing atmosphere (Weyman et al., 2010).

V-nitrogenase has a V-Fe cofactor in the active site. It is encoded by vnfHDGK genes and is expressed in het­erocysts only under Mo-deficient conditions, in the pres­ence of V (Kentemich et al., 1988; Thiel, 1993; Thiel et al., 1995). Biochemical and spectroscopic investigations of purified proteins isolated from Azotobacter vinelandii have revealed a mechanistic difference between the Mo—Fe and V—Fe catalytic site and in H2 evolution mechanisms (Lee et al., 2009).

N2 + 12H+ + 12e~ + 24ATP / 2NH3 + 24ADP + 24P; + 3H2

As can be seen from the Eqns (21.2) and (21.4), the distri­bution of electrons and protons are different for Mo — and V-nitrogenases. V-nitrogenase can produce three
times more H2 per mole of N2 reduced compared to Mo-nitrogenase (Eady, 1996). For this reason, the pro­duction of H2 by vnf system is likely to be more efficient and it is, therefore, worth searching for organisms pos­sessing alternative nitrogenases. For a long time the presence of alternative nitrogenases was confirmed only for A. variabilis and Anabaena azollae (Ni et al., 1990; Thiel,

1993) . Recent screening of 14 different cyanobacterial strains has revealed 8 strains with nif2, and 4 strains with vnf nitrogenases (Masukawa et al., 2009), suggest­ing that alternative hydrogenases are not unique.