Jens Appel

The bidirectional hydrogenase of Synechocystis sp. PCC 6803 works as an electron valve during photosynthesis

Abstract. The activity of the bidirectional hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 was found not to be regulated in parallel to respiration but to photosynthesis. A mutant with a deletion in the large hydrogenase subunit gene (hoxH), which contains the active site, was impaired in the oxidation of photosystem I (PSI) when illuminated with light, which excites either PSI alone or both photosystems. The fluorescence of photosystem II (PSII) of this mutant was higher than that of wild-type cells. The transcript level of the photosynthetic genes psbA, psaA and petB was found to be different in the hydrogenase-free mutant cells compared to wild-type cells, which indicates that the hydrogenase has an effect on the regulation of these genes. Collectively, these results suggest that the bidirectional hydrogenase functions as a valve for low-potential electrons generated during the light reaction of photosynthesis, thus preventing a slowing down of electron transport. This conclusion is supported by growth curves demonstrating that the mutant cells need more time to adapt to changing light intensities. Investigations of the wild-type and ΔhoxH strains strongly suggest that Synechocystis contains only the bidirectional hydrogenase, which seems to be essentially insensitive to oxygen.

Hydrogen metabolism in organisms with oxygenic photosynthesis: hydrogenases as important regulatory devices for a proper redox poising?

Abstract Three different hydrogenases and the nitrogenase putatively participate in the hydrogen metabolism of micro-organisms carrying out oxygenic photosynthesis. Hydrogenases either produce hydrogen or split hydrogen into protons and electrons depending on their redox partners, whereas the nitrogenase produces hydrogen unidirectionally as a byproduct during the reduction of nitrogen to ammonia. Hydrogenases are well-characterized enzymes on the enzymatic, structural and genetic level, especially in prokaryotic micro-organisms. They can be classified regarding the metal composition of their active site (Fe-only, NiFe or metal-free), their preferential direction of reaction (uptake only or bidirectional/reversible) and their in vivo electron donors or acceptors. The main physiological role of the uptake hydrogenase in cyanobacteria is probably recapturing the hydrogen produced by nitrogenase. The role of the bidirectional hydrogenase in phototrophs is still a matter of debate. Based on recent results which showed it to be of the NAD(P)-reducing type, a model for its physiological function is suggested. This model includes that this type of hydrogenase is linked to complex I of the respiratory electron-transport chain and might be an important electron valve during photosynthesis under rapidly changing light conditions. The existence of an Fe-only hydrogenase as well as an NiFe-hydrogenase in green algae is still enigmatic and is discussed as hydrogenases either participating in the production of hydrogen or during fermentation.

Sequence analysis of an operon of a NAD(P)-reducing nickel hydrogenase from the cyanobacterium Synechocystis sp. PCC 6803 gives additional evidence for direct coupling of the enzyme to NAD(P)H-dehydrogenase (complex 1)☆

The sequence of a NAD(P)-reducing hydrogenase operon of Synechocystis sp. PCC 6803 containing genes for a small and a large hydrogenase subunit and six additional ORFs was determined. Until now only 11 of the 14 polypeptides of the NADH-dehydrogenase of E. coli were found in Synechocystis. By sequence homologies we suggest that the missing subunits of the peripheral part of the dehydrogenase, containing most of the FeS-clusters, are encoded by three ORFs of this operon. This hypothesis is discussed in relation to the NAD(P)-reducing hydrogenase of Synechocystis.

LexA regulates the bidirectional hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 as a transcription activator

The bidirectional NiFe‐hydrogenase of Synechocystis sp. PCC 6803 is encoded by five genes (hoxEFUYH) which are transcribed as one unit. The transcription of the hox‐operon is regulated by a promoter situated upstream of hoxE. The transcription start point was located at −168 by 5′Race. Several promoter probe vectors carrying different promoter fragments revealed two regions to be essential for the promoter activity. One is situated in the untranslated 5′leader region and the other is found −569 to −690 nucleotides upstream of the ATG. The region further upstream was shown to bind a protein. Even though an imperfect NtcA binding site was identified, NtcA did not bind to this region. The protein binding to the DNA was purified and found to be LexA by MALDI‐TOF. The complete LexA and its DNA binding domain were overexpressed in Escherichia coli. Both were able to bind to two sites in the examined region in band‐shift‐assays. Accordingly, the hydrogenase activity of a LexA‐depleted mutant was reduced. This is the first report on LexA acting not as a repressor but as a transcriptional activator. Furthermore, LexA is the first transcription factor identified so far for the expression of bidirectional hydrogenases in cyanobacteria.

The hydroxyphenylpyruvate dioxygenase from Synechocystis sp. PCC 6803 is not required for plastoquinone biosynthesis

The disruption of the Synechocystis open reading frame Δslr0090 encoding a gene with high homology to plant genes encoding 4‐hydroxyphenylpyruvate dioxygenase results in an impairment of tocopherol biosynthesis without affecting levels of plastoquinone, carotenoids and chlorophyll as well as cell growth and photosynthesis. Our results indicate that in Synechocystis in contrast to the situation in higher plants the 4‐hydroxyphenylpyruvate dioxygenase is not required for the synthesis of plastoquinone.

Occurrence of hydrogenases in cyanobacteria and anoxygenic photosynthetic bacteria: implications for the phylogenetic origin of cyanobacterial and algal hydrogenases.

Hydrogenases are important enzymes in the energy metabolism of microorganisms. Therefore, they are widespread in prokaryotes. We analyzed the occurrence of hydrogenases in cyanobacteria and deduced a FeFe-hydrogenase in three different heliobacterial strains. This allowed the first phylogenetic analysis of the hydrogenases of all five major groups of photosynthetic bacteria (heliobacteria, green nonsulfur bacteria, green sulfur bacteria, photosynthetic proteobacteria, and cyanobacteria). In the case of both hydrogenases found in cyanobacteria (uptake and bidirectional), the green nonsulfur bacterium Chloroflexus aurantiacus was found to be the closest ancestor. Apart from a close relation between the archaebacterial and the green sulfur bacterial sulfhydrogenase, we could not find any evidence for horizontal gene transfer. Therefore, it would be most parsimonious if a Chloroflexus-like bacterium was the ancestor of Chloroflexus aurantiacus and cyanobacteria. After having transmitted both hydrogenase genes vertically to the different cyanobacterial species, either no, one, or both enzymes were lost, thus producing the current distribution. Our data and the available data from the literature on the occurrence of cyanobacterial hydrogenases show that the cyanobacterial uptake hydrogenase is strictly linked to the occurrence of the nitrogenase. Nevertheless, we did identify a nitrogen-fixing Synechococcus strain without an uptake hydrogenase. Since we could not find genes of a FeFe-hydrogenase in any of the tested cyanobacteria, although strains performing anoxygenic photosynthesis were also included in the analysis, a cyanobacterial origin of the contemporary FeFe-hydrogenase of algal plastids seems unlikely.

Overexpression, Isolation, and Spectroscopic Characterization of the Bidirectional [NiFe] Hydrogenase from Synechocystis sp. PCC 6803

The bidirectional [NiFe] hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 was purified to apparent homogeneity by a single affinity chromatography step using a Synechocystis mutant with a Strep-tag II fused to the C terminus of HoxF. To increase the yield of purified enzyme and to test its overexpression capacity in Synechocystis the psbAII promoter was inserted upstream of the hoxE gene. In addition, the accessory genes (hypF, C, D, E, A, and B) from Nostoc sp. PCC 7120 were expressed under control of the psbAII promoter. The respective strains show higher hydrogenase activities compared with the wild type. For the first time a Fourier transform infrared (FTIR) spectroscopic characterization of a [NiFe] hydrogenase from an oxygenic phototroph is presented, revealing that two cyanides and one carbon monoxide coordinate the iron of the active site. At least four different redox states of the active site were detected during the reversible activation/inactivation. Although these states appear similar to those observed in standard [NiFe] hydrogenases, no paramagnetic nickel state could be detected in the fully oxidized and reduced forms. Electron paramagnetic resonance spectroscopy confirms the presence of several iron-sulfur clusters after reductive activation. One [4Fe4S]+ and at least one [2Fe2S]+ cluster could be identified. Catalytic amounts of NADH or NADPH are sufficient to activate the reaction of this enzyme with hydrogen.

Mutagenesis of hydrogenase accessory genes of Synechocystis sp. PCC 6803

Genes homologous to hydrogenase accessory genes are scattered over the whole genome in the cyanobacterium Synechocystis sp. PCC 6803. Deletion and insertion mutants of hypA1 (slr1675), hypB1 (sll1432), hypC, hypD, hypE and hypF were constructed and showed no hydrogenase activity. Involvement of the respective genes in maturation of the enzyme was confirmed by complementation. Deletion of the additional homologues hypA2 (sll1078) and hypB2 (sll1079) had no effect on hydrogenase activity. Thus, hypA1 and hypB1 are specific for hydrogenase maturation. We suggest that hypA2 and hypB2 are involved in a different metal insertion process. The hydrogenase activity of ΔhypA1 and ΔhypB1 could be increased by the addition of nickel, suggesting that HypA1 and HypB1 are involved in the insertion of nickel into the active site of the enzyme. The urease activity of all the hypA and hypB single‐ and double‐mutants was the same as in wild‐type cells. Therefore, there seems to be no common function for these two hyp genes in hydrogenase and urease maturation in Synechocystis. Similarity searches in the whole genome yielded Slr1876 as the best candidate for the hydrogenase‐specific protease. The respective deletion mutant had no hydrogenase activity. Deletion of hupE had no effect on hydrogenase activity but resulted in a mutant unable to grow in a medium containing the metal chelator nitrilotriacetate. Growth was resumed upon the addition of cobalt or methionine. Because the latter is synthesized by a cobalt‐requiring enzyme in Synechocystis, HupE is a good candidate for a cobalt transporter in cyanobacteria.

Protection by α-tocopherol of the repair of photosystem II during photoinhibition in Synechocystis sp. PCC 6803

α-Tocopherol is a lipophilic antioxidant that is an efficient scavenger of singlet oxygen. We investigated the role of α-tocopherol in the protection of photosystem II (PSII) from photoinhibition using a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that is deficient in the biosynthesis of α-tocopherol. The activity of PSII in mutant cells was more sensitive to inactivation by strong light than that in wild-type cells, indicating that lack of α-tocopherol enhances the extent of photoinhibition. However, the rate of photodamage to PSII, as measured in the presence of chloramphenicol, which blocks the repair of PSII, did not differ between the two lines of cells. By contrast, the repair of PSII from photodamage was suppressed in mutant cells. Addition of α-tocopherol to cultures of mutant cells returned the extent of photoinhibition to that in wild-type cells, without any effect on photodamage. The synthesis de novo of various proteins, including the D1 protein that plays a central role in the repair of PSII, was suppressed in mutant cells under strong light. These observations suggest that α-tocopherol promotes the repair of photodamaged PSII by protecting the synthesis de novo of the proteins that are required for recovery from inhibition by singlet oxygen.

The Entner–Doudoroff pathway is an overlooked glycolytic route in cyanobacteria and plants

Significance Life on Earth is substantially driven by a circuit of photosynthesis and glucose oxidation. Photosynthesizers capture sunlight and store its energy in the bonds of carbohydrates. Oxidation of carbohydrates provides organisms with a source of ATP and organic carbon for the synthesis of cellular building blocks. Our data provide strong evidence that the Entner–Doudoroff pathway of glucose degradation, which has been previously long overlooked, operates in cyanobacteria and plants. Phylogenetic analyses reveal that the cyanobacterial ancestor of plastids transferred this glycolytic route, via endosymbiotic gene transfer, to the plant lineage. Glucose degradation pathways are central for energy and carbon metabolism throughout all domains of life. They provide ATP, NAD(P)H, and biosynthetic precursors for amino acids, nucleotides, and fatty acids. It is general knowledge that cyanobacteria and plants oxidize carbohydrates via glycolysis [the Embden–Meyerhof–Parnas (EMP) pathway] and the oxidative pentose phosphate (OPP) pathway. However, we found that both possess a third, previously overlooked pathway of glucose breakdown: the Entner–Doudoroff (ED) pathway. Its key enzyme, 2-keto-3-deoxygluconate-6-phosphate (KDPG) aldolase, is widespread in cyanobacteria, moss, fern, algae, and plants and is even more common among cyanobacteria than phosphofructokinase (PFK), the key enzyme of the EMP pathway. Active KDPG aldolases from the cyanobacterium Synechocystis and the plant barley (Hordeum vulgare) were biochemically characterized in vitro. KDPG, a metabolite unique to the ED pathway, was detected in both in vivo, indicating an active ED pathway. Phylogenetic analyses revealed that photosynthetic eukaryotes acquired KDPG aldolase from the cyanobacterial ancestors of plastids via endosymbiotic gene transfer. Several Synechocystis mutants in which key enzymes of all three glucose degradation pathways were knocked out indicate that the ED pathway is physiologically significant, especially under mixotrophic conditions (light and glucose) and under autotrophic conditions in a day/night cycle, which is probably the most common condition encountered in nature. The ED pathway has lower protein costs and ATP yields than the EMP pathway, in line with the observation that oxygenic photosynthesizers are nutrient-limited, rather than ATP-limited. Furthermore, the ED pathway does not generate futile cycles in organisms that fix CO2 via the Calvin–Benson cycle.