ABSTRACTHydrogenases catalysed production of biological hydrogen provides an alternative and cleanway of producing energy over the existing physico-chemical methods. This review placesemphasis on hydrogenases, a group of enzymes that catalyses H2 formation from protons oroxidation protons. The three categories of hydrogenases (i.e. NiFe-, FeFe-, and Fe-hydrogenases) and characteristics of these enzymes which are vital in the understanding of themechanism of H2 production, the control of cell metabolism and increase in the yield of H2production are also discussed. Biological hydrogen production is grouped based on light energyrequirement and feedstock sources. Other applicable forms of hydrogenases are also discussed.Key words: HydrogenasesBiological Hydrogen ProductionFermentationHYDROGENASES CATALISED PRODUCTION OF BIOLOGICAL HYDROGENOur lifestyle as humans are influenced and sustained by energy. As a society, our energy sectorhas undergone development from the use of wood to coal, petroleum, wind, natural gas, solarand nuclear. For decades, we have discovered the need to be efficient in the use of energy.Due to the current usage of fossil fuel which will plunge the world into energy crises, it isnecessary to employ a cleaner energy production source devoid of environmental pollution andtowards the sustainable development goals. These energy sources when patronized, wouldreduce environmental impact and therefore enhance the energy security of the world at large.Among the various sources of sustainable energy dihydrogen is regarded, as it is the mostsustainable energy source since it has higher energy content of about 2.75 percent compared tohydrocarbon fuels(gasoline) and produces only water upon combustion (Kim and Kim, 2011).According to (Kim and Kim, 2011), dihydrogen can be used in fuel cells producing electricitywith higher efficiency.Primarily the production of dihydrogen for the past years has been because of steamreformation of hydrocarbons and coal gasification, which originates from fossil fuels andrequires high temperature and pressure conditions. Meanwhile, there is an environmentallyfriendly way of producing H2 biologically.Biological production of dihydrogen is dependent on an H2 metabolizing enzyme. H2 producingenzymes catalyses the simplest chemical reaction 2H+ + 2e H2.Studies have shown that the active enzyme units are synthesized in a complex processinvolving auxiliary enzymes and protein maturation steps. Currently, three enzymes areinvolved in dihydrogen production namely: Fe-Fe hydrogenase, Fe hydrogenase and Ni-Fehydrogenases.NiFe-HYDROGENASESNiFe hydrogenases is the largest group of hydrogenases known. It is considered that Nickelcontaining hydrogenases are less sensitive than Fe-Fe hydrogenases to the inhibition of COand O2. The structure of the enzyme is made up of an ?, ? heterodimer; with the ?- subunitbeing larger one and contains biometallic active site, whereas the small ?-subunit possessesFe-S clusters, which transfers electrons between the active sites and the electron acceptor ordonor binding site. According to the diagram below the biometallic NiFe is bound to the Satoms of four cysteine residues. In addition, non protenous ligands, one CO and two CN arecoordinated to the Fe atom (Happe et al., 1997).With respect to the small and large subunits, NiFe hydrogenases have been characterized intofour groups in relation to the function of the enzymes.GROUP I represents the NiFe uptake hydrogenases which are mostly found in Wolinellasuccinogenes, Aquifex aeolicus, Thiocapsa roseopersicina, and some Desulfovibrio sp. Theseare membrane bound enzymes which attribute the reduction of dihydrogen to the presence ofanaerobic electron acceptors such as NO3-, SO42-, fumarate, or CO2 (anaerobic respiration) orto O2 (aerobic respiration), with the recovery of energy in the form of a proton-motive force.In relation to structure, these enzymes are classified according to the presence of a long signalpeptide at the N terminus of their small subunit (Sargent et al., 2006). The signal peptide servesas a signal peptide targeting the fully folded heterodimer to the membrane and periplasm.GROUP II comprises of cytoplasmic dihydrogen sensors and cynobacteria uptake nickel ironhydrogenases. It does not contain a signal peptide. Cytoplasmic dihydrogen sensors remain inthe cytoplasm and detects the presence of dihydrogen in the environment and trigger manychemical reactions controlling the synthesis of hydrogenases (Kleihues et al., 2000). This typeof hydrogenase has been found in Bradyrhizobium japonicum, Rhodobacter eutropha,Rhodobacter capsulatus, and Rhodopseudomonas palustris.Cynobacteria hydrogenases consists of Nostoc and Anabaena variabilis. These are stimulatedunder the influence of nitrogen fixation conditions and localized on the cytoplasm side of eitherthe cytoplasmic or thylakoid membrane (Kim & Kim, 2011).Bidirectional heteromultimeric cytoplasmic NiFe-hydrogenases also known as group threefunctions reversibly and reoxidize the cofactors such as 420 (F420, 8-hydroxy-5deazaflavin),NAD, or NADP. Many members of this group are found in archea such asMethanothermobacter marburgensis and Methanosarcina mazei, which function to reduce S0to H2S, use NADPH as electron donor, and provide reducing equivalents for heterodisulfidereductase ( Ma et al., 2000).GROUP III also known as bidirectional heteromultimeric cytoplasmic hydrogenases are foundin archea such Methanothermobacter marbugensis and Methanosarcina mazei, whichfunctions to reduce S0 to H2S, which uses NADPH as electron donor, and provide reducingequivalents for heterodisulphide. This group of hydrogenases function reversibly by oxidizingthe cofactors such as cofactor 420 ( F420 8-hydroxy-5-5- deazaflavin) and other subunits thatare able to bind cofactors ( Ma et al., 2000).GROUP IV also comprises of dihydrogen evolution, energy conserving membrane associatedhydrogenases which constitutes of six or more subunits. Majority of these enzymes have beenfound in archea, including methanosarcina bakeri, Methanothermobacter marburgensis, andpyrococcus furiosus. They aid the formation of dihydrogen by reducing ferredoxin, which isgenerated by the oxidation of the carbonyl group of acetate to CO2 (Kunkel et al., 1998).According to (Sauter et al., 1992; Fox et al., 1996) group IV hydrogenases are also found inE.coli and R. rubrum, which reduce protons from water in order to dispose of the excessreducing equivalents produced by the anaerobic oxidation of organic compounds of lowpotential such as CO or formate.FeFe HydrogenasesContrary to NiFe hydrogenases made up of at least two subunits many FeFe hydrogenasesare monomeric in nature and contain only a catalytic subunit and differs considerably in size.Fig (a) Active site of NiFehydrogenasesAside the conservation of ca 350 residues containing the active site cluster(H-cluster), thereare additional domains which houses Fe-S clusters. According (Adams, 1990), the H clusterconsists of a binuclear FeFe bound to 4Fe-4S by bridging cysteine and attached to theprotein by four cysteine ligands. Ligands such as CO and CN are attached to Fe atoms and twobridging sulphur atoms are coordinated with Fe atoms (Fig b).Anaerobic prokaryotes such as clostridia and sulphate reducers have proven to a source ofFeFe hydrogenases and in eukaryotes ( Atta and Meyer, 2000; Horner et al., 2000). These arethe only source of hydrogenases for eukaryotes (Vignais and Colbeau, 2004). Generally NiFehydrogenases are involved in dihydrogen consumption whiles FeFe hydrogenases areinvolved in dihydrogen production. However it is also believed to act as uptake hydrogenasein D. Vulgaris, a formate dehydrogenase in Eubacterium acidaminophilum, and an electron”valve” that enhances the suvival of the algae under anaerobic conditions (Kim & Kim, 2011).Fe HydrogenaseIt was first discovered in methanothermobactermaburgenis which catalysis carbon dioxidereduction with dihydrogen to methane (Vignais and Billoud, 2007). With the absence of FeS clusters or nickel it was named metal-free hydrogenases. This hydrogenase differs from theFig (b) : active site of FeFehydrogenasesother two hydrogenases NiFe and FeFe hydrogenases not only structurally but with respectto the redox inactiveness of the Fe required for enzymatic activity. They do not catalyse2H+ + 2e- H2 reversible redox reaction.BIOLOGICAL HYDROGEN PRODUCTIONIt is a novel area in chemistry which employs microorganisms that freely and efficientlyproduce dihydrogen as a by-product of metabolism. Solar energy or electrons contained inorganic and inorganic molecules can produce dihydrogen (McKinlay & Harwood, 2010).Microorganisms produce dihydrogen for two main reasons. First to dispose off excess reducingfermentation processes that is carried out in a dark anaerobic process or associated with anoxicphotosynthetic activity. Also, H2 is the byproduct of the activities of nitrogenase, the enzymeresponsible for the fixation of nitrogen in the atmosphere. BHP can be broadly split into photodriven and dark processes depending on the light energy requirement. It can further be spitaccording to feedstock sources.Direct biophotolysisWater is decomposed into H2 and O2 with the aid of light. This is by far the simplest reactionFig C: active site of Fehydrogenaseswhich involves only water and light and it evolves no greenhouse gases. This method is theultimate way of producing H2 (Zaborsky, 1998).2H2O light energy 2H2 +O2 ?G = 237kJStudies have shown that organisms in nature have demonstrated low rates of H2 productiondue to large energy barrier needed to overcome (+237KJ/mol H2 ). Direct biophotolysis is avery attractive method to produce H2 but the main challenge is the suppression of hydrogenaseby the byproduct O2.Indirect biophotolysisIntensive research has focused on decreasing the sensitivity of O2 through indirectbiophotolysis in which dihydrogen production is spatially or temporally separated fromphotosynthesis (Turner et al., 2008). Through this method, glycogen and starch accumulatedduring carbon dioxide fixation is degraded to produce H2 by an anaerobic process. This usuallyhappens in the dark or in the light condition with cells with impaired O2 evolving photosystems.The levels of O2 is kept low by specialized cynobacterial cells known as heterocysts (Sakuraiand Masukawa, 2008).Photo-fermentationPhoto-fermentation is the fermentative conversion of organic substrate to biohydrogenmanifested via some diverse forms of photosynthetic bacteria through a sequence ofbiochemical reactions comparable to anaerobic conversion. Photo-fermentation differs fromdark fermentation because it only proceeds in the presence of light. Photo-fermentation bacteriaobtain electrons from organic compounds such as acetate, butyrate, lactate and so forth orthrough inorganics such as S2O3 2-, H2S, Fe2+ (Mckinlay and Harwood, 2010). These obtainedelectrons are finally released as H2 through the action nitrogenases (Koku et al., 2002). Photofermentation is the most efficient of all methods for the production dihydrogen theoreticallybecause of its application to numerous organic substrates and can also be applied in the fieldof water treatment. On the contrary, this process consumes a lot of energy and about 30% ofthe electrons are contributed to biomass growth (Honda et al., 2006). It additionally requires anitrogen-restricting surroundings, but normally, real waste carries nitrogen above the levelsthat hinders H2 generating mechanism. Also, the very last H2 yield can be reduced with the aidof the uptake hydrogenase activity (Kars and Gunduz, 2010).Dark FermentationThe fermentative conversion of an organic substrate into biohydrogen without the use of lightis called dark fermentation. Fermentative bacteria such as Enterobacter sp., Bacillus sp., andClostridium sp. can produce H2 from carbohydrate containing substrates in a dark environment.However, most of the studies has been focused on clostridium sp. because of its highest H2production (1.61–2.36 mol H2/mol glucose) was obtained for them and they are abundant innature (Hawkers et al., 2002). H2 production under this process is the fastest and can yield ashigh as 15 L H2/Lreactor/h which is 100 times higher than the photo-fermentation process (Wuet al., 2005). It is also effective in the treatment of waste such as food waste, sewage sludgeand lignocellilosic waste as a means of supplying energy and protecting the environment.CO gas fermentationCO gas has been of interest in the production of H2 because of the ability of the gas to convertcarbon-rich materials in favour of CO. Despite the slowness of biological processes, theypossess several advantages including higher specificity, higher yields, lower energy costs andthe ability to resist poisoning (Bredwell et al., 1999). Despite the little knowledge known aboutthe mechanism of CO gas fermentation process, it is clearly stated that carbon monoxidedehydrogenase is the major contribution to this mechanism. Also, coupled with the carbonmonoxide dehydrogenase are two other enzymes; ferredoxin-like cofactor (Fe-S proteins) anda terminal hydrogenase that produces hydrogenase at a high rate involved in the oxidation ofCO to H2. Carbon monoxide hydrogenase is responsible for the oxidation of CO to CO2 andreduction equivalents. The reduction equivalents are then transferred through the Fe-S proteincofactor, to a hydrogenase to produce H2 (Klasson et al 1992). The production of H2 occurs intwo consecutive reactions through the oxidation and reduction processes. The oxidation and ofCO in an anaerobic process with Fd in the equation below.Application of hydrogenases in other fieldsHydrogenases are very versatile molecules despite its limit to the production of H2 accordingto this research. Its application in chemistry is enormous with its use ranging fromelectrocatalyst, bioremediation and for the synthesis of fine chemicals. Hydrogenases frombacteria such as Thiocapsa roseopersicina has attracted much interest due to its high productionof H2 and strong activity and stability on denaturing agents such as oxygen and temperature.ConclusionThere is a higher probability that the future of sustainable and clean energy will depend on thedevelopment of biological hydrogen production technologies as it solely depends on naturalsunlight and water as well as abundant biomass. H2 promises remarkable potential for settlingglobal energy challenges not just in a small way, but substitute for an environmentally friendlyenergy source despite the challenges associated with it.ariculture-r6.