Occurrence, classification, and biological function of hydrogenases: an overview.

Hydrogenases (H2ases) catalyze the reversible oxidation of molecular hydrogen and play a central role in microbial energy metabolism. Most of these enzymes are found in Archaea and Bacteria, but a few are present in Eucarya as well. They can be distributed into three classes: the [Fe]-H2ases, the [NiFe]-H2ases, and the metal-free H2ases. The vast majority of known H2ases belong to the first two classes, and over 100 of these enzymes have been characterized genetically and/or biochemically. Compelling evidence from sequences and structures indicates that the [NiFe]- and [Fe]-H2ases are phylogenetically distinct classes of proteins. The catalytic core of the [NiFe]-H2ases is a heterodimeric protein, although additional subunits are present in many of these enzymes. Functional classes of [NiFe]-H2ases have been defined, and they are consistent with categories defined by sequence similarity of the catalytic subunits. The catalytic core of the [Fe]-H2ases is a ca. 350-residue domain that accommodates the active site (H-cluster). A few monomeric [Fe]-H2ases are barely larger than the H-cluster domain. Many others are monomeric as well, but possess additional domains that contain redox centers, mostly iron–sulfur. Some [Fe]-H2ases are oligomeric. The modular structure of H2ases is strikingly illustrated in recently unveiled sequences and structures. It is also remarkable that most of the accessory domains and subunits of H2ases have counterparts in other redox complexes, in particular NADH-ubiquinone oxidoreductase (Complex I) of respiratory chains. Microbial genome sequences are bringing forth a significant body of additional H2ase sequence data and contribute to the understanding of H2ase distribution and evolution. Altogether, the available data suggest that [Fe]-H2ases are restricted to Bacteria and Eucarya, while [NiFe]-H2ases, with one possible exception, seem to be present only in Archaea and Bacteria. H2ase processing and maturation involve the products of several genes which have been identified and are currently being characterized in the case of the [NiFe]-H2ases. In contrast, near to nothing is known regarding the maturation of the [Fe]-H2ases. Inspection of the currently available genome sequences suggests that the [NiFe]-H2ase maturation proteins have no similar counterparts in the genomes of organisms possessing [Fe]-H2ases only. This observation, if confirmed, would be consistent with the phylogenetic distinctiveness of the two classes of H2ases. Sequence alignments of catalytic subunits of H2ases have been implemented to construct phylogenetic trees that were found to be consistent, in the main, with trees derived from other data. On the basis of the comparisons performed and discussed here, proposals are made to simplify and rationalize the nomenclature of H2ase-encoding genes.

Classification and phylogeny of hydrogenases.

The U.S. Department of Energy and the National Renewable Energy Laboratory are developing technologies to produce hydrogen from renewable, sustainable sources. A cost goal of $2.00–$3.00 kg−1 of hydrogen has been identified as the range at which delivered hydrogen becomes cost competitive with gasoline for passenger vehicles. Electrolysis of water is a standard commercial technology for producing hydrogen. Using wind and solar resources to produce the electricity for the process creates a renewable system. Biomass-to-hydrogen processes, including gasification, pyrolysis, and fermentation, are less well-developed technologies. These processes offer the possibility of producing hydrogen from energy crops and from biomass materials such as forest residue and municipal sewage. Solar energy can be used to produce hydrogen from water and biomass by several conversion pathways. Concentrated solar energy can generate high temperatures at which thermochemical reactions can be used to split water. Photoelectrochemical water splitting and photobiology are long-term options for producing hydrogen from water using solar energy. All these technologies are in the development stage. Copyright © 2007 John Wiley & Sons, Ltd.

Renewable hydrogen production

Global climate change has stimulated efforts to reduce CO(2) emissions. One approach to addressing this problem is to recycle CO(2) directly into fuels or chemicals using photosynthesis. Here we genetically engineered Synechococcus elongatus PCC7942 to produce isobutyraldehyde and isobutanol directly from CO(2) and increased productivity by overexpression of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). Isobutyraldehyde is a precursor for the synthesis of other chemicals, and isobutanol can be used as a gasoline substitute. The high vapor pressure of isobutyraldehyde allows in situ product recovery and reduces product toxicity. The engineered strain remained active for 8 d and produced isobutyraldehyde at a higher rate than those reported for ethanol, hydrogen or lipid production by cyanobacteria or algae. These results underscore the promise of direct bioconversion of CO(2) into fuels and chemicals, which bypasses the need for deconstruction of biomass.

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Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde

Investigating and Exploiting the Electrocatalytic Properties of Hydrogenases

Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strains have been investigated on the molecular and the physiological level in order to find the most efficient organisms for photobiological hydrogen production. These strains were screened for the presence or absence of hup and hox genes, and it was shown that they have different sets of genes involved in H2 evolution. The uptake hydrogenase was identified in all N2-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme. In N2-fixing strains, hydrogen was mainly produced by the nitrogenase as a by-product during the reduction of atmospheric nitrogen to ammonia. Therefore, hydrogen production was investigated both under non-nitrogen-fixing conditions and under nitrogen limitation. It was shown that the hydrogen uptake activity is linked to the nitrogenase activity, whereas the hydrogen evolution activity of the bidirectional hydrogenase is not dependent or even related to diazotrophic growth conditions. With regard to large-scale hydrogen evolution by N2-fixing cyanobacteria, hydrogen uptake-deficient mutants have to be used because of their inability to re-oxidize the hydrogen produced by the nitrogenase. On the other hand, fermentative H2 production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.

Cyanobacterial H2 production - a comparative analysis

A 10-kb DNA region of the cyanobacterium Anabaena variabilis ATCC 29413 containing the structural genes of the uptake hydrogenase ( hupSL ) was cloned and sequenced. In contrast to the hupL gene of Anabaena sp. strain PCC 7120, which is interrupted by a 10.5-kb DNA fragment in vegetative cells, there is no programmed rearrangement within the hupL gene during the heterocyst differentiation of A. variabilis . The hupSL genes were transcribed as a 2.7-kb operon and were induced only under nitrogen-fixing conditions, as shown by Northern blot experiments and reverse transcriptase PCR. Primer extension experiments with a fluorescence-labeled oligonucleotide primer confirmed these results and identified the 5′ start of the mRNA transcript 103 bp upstream of the ATG initiation codon. A consensus sequence in the promoter that is recognized by the fumarate nitrate reductase regulator (Fnr) could be detected. The hupSL operon in A. variabilis was interrupted by an interposon deletion (mutant strain AVM13). Under N 2 -fixing conditions, the mutant strain exhibited significantly increased rates in H 2 accumulation and produced three times more hydrogen than the wild type. These results indicate that the uptake hydrogenase is catalytically active in the wild type and that the enzyme reoxidizes the H 2 developed by the nitrogenase. The Nif phenotype of the mutant strain showed a slight decrease of acetylene reduction compared to that of the wild type.

Kathrin Schütz

Papers

Cyanobacterial H2 production - a comparative analysis

Transcriptional and mutational analysis of the uptake hydrogenase of the filamentous cyanobacterium Anabaena variabilis ATCC 29413.

HoxE—a subunit specific for the pentameric bidirectional hydrogenase complex (HoxEFUYH) of cyanobacteria

A hydrogen-producing, hydrogenase-free mutant strain of Nostoc punctiforme ATCC 29133

Molecular cloning and characterization of hetR genes from filamentous cyanobacteria