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.The only microorganisms with an O 2 -producing photosynthesis that have a hydrogen metabolism are cyanobacteria and green algae (23,25). In cyanobacteria, up to three enzymes can be involved in hydrogen metabolism: the nitrogenase which produces H 2 during nitrogen fixation (24), the membranebound hydrogenase which reoxidizes the H 2 (10), and the bidirectional hydrogenase catalyzing both oxidation of molecular hydrogen and reduction of protons (38). In cyanobacteria, the genetics of bidirectional hydrogenases are especially well characterized (3, 7). But, the H 2 production in filamentous cyanobacteria during the reduction of nitrogen to NH 3 is mainly catalyzed by the nitrogenase in the heterocysts. The Anabaena cells can oxidize the hydrogen with the uptake hydrogenase via the oxyhydrogen (Knallgas) reaction. It was suggested that the organism gets additional ATP while the Knallgas reaction can protect the O 2 -sensitive nitrogenase by removing the oxygen in the heterocysts (30).Hydrogenases have been described for a large number of microorganisms and studied intensively in diverse phylogenetic groups of bacteria (22,34,54). The uptake hydrogenases are membrane-bound enzymes which consist of two subunits with [Fe-S] clusters as prosthetic groups. The large subunit, HupL, carries additionally a Ni atom in the active center. In most of the bacterial families, the hupSL genes are clustered in an operon in which hupS is located upstream of the hupL gene (20,51,53). Recently, some hydrogenase sequences from filamentous cyanobacteria were published (10, 31, 38). The uptake hydrogenase i...

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 H(2) evolution. The uptake hydrogenase was identified in all N(2)-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme. In N(2)-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 N(2)-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 H(2) production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.

NAD(P)(+)-reducing hydrogenases have been described to be composed of a diaphorase (HoxFU) and a hydrogenase (HoxYH) moiety. This study presents for the first time experimental evidence that in cyanobacteria, a fifth subunit, HoxE, is part of this bidirectional hydrogenase. HoxE exhibits sequence identities to NuoE of respiratory complex I of Escherichia coli. The subunit composition of the cyanobacterial bidirectional hydrogenase has been investigated. The oxygen labile enzyme complex was purified to close homogeneity under anaerobic conditions from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 6301. The 647-fold and 1290-fold enriched purified enzyme has a specific activity of 46 micromol H(2) evolved (min mg protein)(-1) and 15 micromol H(2) evolved (min mg protein)(-1), respectively. H(2)-evolution of the purified enzyme of S. sp. PCC 6803 is highest at 60 degrees C and pH 6.3. Immunoblot experiments, using a polyclonal anti-HoxE antibody, demonstrate that HoxE co-purifies with the hydrogenase activity in S. sp. PCC 6301. SDS-PAGE gels of the purified enzymes revealed six proteins, which were partially sequenced and identified, besides one nonhydrogenase component, as HoxF, HoxU, HoxY, HoxH and, remarkably, HoxE. The molecular weight of the native protein (375 kDa) indicates a dimeric assembly of the enzyme complex, Hox(EFUYH)(2).

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