Hydrogenases in Green Algae and Photobiological Production of H2 Gas

Hydrogenases are a class of enzymes that catalyze the reversible reduction of protons (H+) to molecular hydrogen (H2). The recent demonstration that the green alga Scenedesmus obliquus generates large quantities of hydrogen gas upon illumination has created interest in photobiological (H2) production, as hydrogen gas may be an alternative energy source in the future. The interest stems from the fact that (H2) is a clean source of energy; unlike fossil fuels, it does not generate carbon dioxide upon burning. In addition, (H2) fuel is potentially a renewable resource, if technology for mass production of (H2) using algal cultures is perfected.

In a review article published in the June, 2002 issue of Trends in Plant Science (vol 7: 246-250), Thomas Happe, Anja Hemschemeter, Martin Winkler and Annette Kaminski (Botanische Institut, Universitat Bonn, Germany) discuss the latest research on algal hydrogen metabolism pose the question of whether bioproduction of (H2) could help to solve our energy problems in the foreseeable future.

Hydrogenases are dividided into three classes based on the metal composition of their catalytic center: [NiFe]-hydrogenases, [Fe]-hydrogenases and metal-free hydrogenases. [Fe]-hydrogenases have a unique prosthetic group named the H-cluster in their catalytic site, and thus have 100-fold higher activity than hydrogenases in the other two groups. Over 20 genes encoding [Fe]-hydrogenases have been identified in recent years, isolated from hydrogen-producing anaerobic bacteria and several eukaryotes that use protons as terminal electron acceptors under anaerobic conditions. [Fe]-hydrogenases are further divided into three families based upon the number and type of prosthetic [Fe-S] clusters, called F-clusters, and upon the type of natural electron donor. All the three [Fe]-hyrogenase families share three highly conserved domains containing the four cysteines that coordinate the active center. The first family of [Fe]-hydrogenases consists exclusively of enzymes found in green algae. They are monomeric proteins referred to as HydA and have a truncated N-terminus compared to the other two families of [Fe]-hydrogenases. The HydA enzymes have a high specific activity of about 1000 units per mg protein. In green algae, ferridoxin (PetF) serves as the physiological electron donor to HydA, tranferring electrons from the photosynthetic electron transport chain to the chloroplastic HydA enzyme under anaerobic conditions. HydA genes have been identified in Chlorococcum littoraleChlorella fuscaS. obliquus and Chlamydomonas reinhardtii. However, HydA is not universally present in all green algae.

HydA-like sequences have been identified in aerobic eukaryotes, including Arabidopsis. The HydA-like proteins share three protein domains with corresponding [Fe]-hydrogenases. A gene encoding one such protein is called NARF (nuclear prelamin recognition factor), and was first identified in humans as being involved in assembly of nuclear lamina. NARF is not a functional hydrogenase, possibly because it lacks a cysteine residue required for proton transfer in [Fe]-hydrogenases. In yeast, NARF has been shown to be essential, despite the fact that yeast lack prolamin A. Arabidopsis also possesses a NARF-like gene that encodes a protein of 474 amino acids, but its biological significance is unknown.

Fermentative metabolism of photosynthetic eukaryotes
In green algae, (H2) is generated as a result of either anaerobiosis or nutrient deprivation such as sulfur deficiency. Photosynthetic algae sometimes encounter anoxia in their natural habitats due to lack of circulation in water bodies such as ponds, lakes, or sea sediments or owing to oxygen deprivation during algal blooms.

The authors discuss two metabolic pathways operating during fermentation – one pathway under dark conditions and the other under light conditions. The algal [Fe]-hydrogenases has its main role during fermentation that occurs in the light, resulting in the production of large quantities of H2, formate and carbon doxide while reducing the production of ethanol and eliminating totally the production of acetate. Thus, hydrogenase participates in reorganization of reducing equivalents and photosynthetically-generated electrons from fermentation by reduction of protons. H2 gas ultimately acts as the major electron sink under these conditions.

The fermentative pathways in green algae are more efficient than in higher plants. Even higher plant species known for their tolerance to oxygen deprivation have limited survival capacity, indicating that anaerobic metabolism is a suitable substitute for aerobic metabolism for only a short period of time in higher plants. Maize roots, for example, survive only for three days in an oxygen deficient environment.

Hydrogenases under nutrient stress conditions
It has been demonstrated that under conditions of oxygen shortage, Chlamydomonas reinhardtii cells switch to fermentative metabolism within minutes. C. reinhardtii cultures produce large amounts of H2 gas when experiencing sulfur deprivation, and although the conditions of nutrient stress cannot be examined independently of anoxic conditions, the authors describe metabolic events that lead to the enhanced production of hydrogen. When nutrients such as sulfur are severely limiting, the operation of the Photosystem II and thus production of oxygen slows. Respiration continues to consume oxygen and the photosystem I pathway does not slow greatly. Electrons released from the degradation of starch, proteins or lipids can be fed into the plastoquinone pool by NADPH reductase. Electrons are delivered to [Fe]-hydrogenases, leading to abundant hydrogen gas production. The authors state that the hydrogenase thus acts as an electron valve under these conditions to prevent oxidative damage to cell components.

Perspectives
The recent demonstration of the production of 1-2 liters of H2 gas per day by a 10-liter culture of green algae opens up a new chapter in the biofuel technology, necessitating further research in this area. Before scaling up laboratory results into pilot plant projects, three main problems need to be addressed to for large-scale production of H2 gas by algal cultures. These are: (1) elevating hydrogenase levels in the algal cells; (b) reducing the oxygen sensitivity of the enzyme and (c) maximizing the photosynthetic efficiency. The authors suggest that a study of the catalytic principle of hydrogenases may help d

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