Sustained Photoevolution of Molecular Hydrogen in a Mutant of Synechocystis sp. Strain PCC 6803 Deficient in the Type I NADPH-Dehydrogenase Complex

Abstract

The interaction between hydrogen metabolism, respiration, and photosynthesis was studied in vivo in whole cells of Synechocystis sp. strain PCC 6803 by continuously monitoring the changes in gas concentrations (H 2 , CO 2 , and O 2 ) with an online mass spectrometer. The in vivo activity of the bidirectional [NiFe]hydrogenase [H 2 :NAD(P) oxidoreductase], encoded by the hoxEFUYH genes, was also measured independently by the protondeuterium (H-D) exchange reaction in the presence of D 2 . This technique allowed us to demonstrate that the hydrogenase was insensitive to light, was reversibly inactivated by O 2 , and could be quickly reactivated by NADH or NADPH (؉H 2 ). H 2 was evolved by cells incubated anaerobically in the dark, after an adaptation period. This dark H 2 evolution was enhanced by exogenously added glucose and resulted from the oxidation of NAD(P)H produced by fermentation reactions. Upon illumination, a short (less than 30-s) burst of H 2 output was observed, followed by rapid H 2 uptake and a concomitant decrease in CO 2 concentration in the cyanobacterial cell suspension. Uptake of both H 2 and CO 2 was linked to photosynthetic electron transport in the thylakoids. In the ndhB mutant M55, which is defective in the type I NADPH-dehydrogenase complex (NDH-1) and produces only low amounts of O 2 in the light, H 2 uptake was negligible during dark-to-light transitions, allowing several minutes of continuous H 2 production. A sustained rate of photoevolution of H 2 corresponding to 6 mol of H 2 mg of chlorophyll ؊1 h ؊1 or 2 ml of H 2 liter ؊1 h ؊1 was observed over a longer time period in the presence of glucose and was slightly enhanced by the addition of the O 2 scavenger glucose oxidase. By the use of the inhibitors DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] and DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), it was shown that two pathways of electron supply for H 2 production operate in M55, namely photolysis of water at the level of photosystem II and carbohydrate-mediated reduction of the plastoquinone pool.Cyanobacteria are oxygenic photosynthetic prokaryotes containing two photosystems, PS II and PS I, linked by a linear electron transport chain similar to that of higher plant or algal chloroplasts. Much attention has been focussed on H 2 -producing cyanobacteria in the hope that the photosynthetic reactions producing reductant from water can be coupled to the reduction of protons to generate molecular hydrogen at the expense of solar energy (3,11,24,31,32,39). In cyanobacteria, two different enzymes capable of H 2 production in vivo have been described, nitrogenase and reversible hydrogenase. The first enzyme, which occurs only in N 2 -fixing strains, reduces protons to H 2 concomitantly with the reduction of N 2 to NH 4 ϩ . In these organisms, however, H 2 production is limited due to an efficient recycling of H 2 by an uptake [NiFe]hydrogenase, which is present in all known N 2 -fixing strains (31, 39). Accordingly, it has been demonstrated that mutations impairing the activi...

Experimental approaches to kinetics of gas diffusion in hydrogenase

“…The isotope-exchange assay is described in ref. 28. The electrochemical and spectroscopic characterizations were carried out as in refs.…”

“…Hydrogenase activity can be assayed by using mass spectrometry to measure the rates of D 2 , HD, and H 2 exchange in the absence of a redox partner (28). Fig.…”

Integrating dark and light bio-hydrogen production strategies: towards the hydrogen economy

“…However, a mutant of Synechocystis deficient in uptake hydrogenase activity photoproduced H 2 at a rate of 6 μmol H 2 /mg chlorophyll a/h (2 mL/L/h) (Cournac et al 2004). …”

Induction of Photosynthetic Carbon Fixation in Anoxia Relies on Hydrogenase Activity and Proton-Gradient Regulation-Like1-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii

“…At light onset after a prolonged period in dark anoxic conditions, the photosynthetic electron flow is mainly a LEF toward hydrogenase , and lack of hydrogenase activity in hydrogenase maturation factor EF (hydEF) mutant strain deficient in hydrogenases maturation (Posewitz et al, 2004) induces a lag in induction of PSII activity . In cyanobacteria, the bidirectional Ni-Fe hydrogenase might also work as an electron valve for disposal of electrons generated at the onset of illumination of cells (Cournac et al, 2004) or when excess electrons are generated during photosynthesis, preventing the slowing of the electron transport chain under stress conditions (Appel et al, 2000;Carrieri et al, 2011). The bidirectional Ni-Fe hydrogenase could also dispose of excess of reducing equivalents during fermentation in dark anaerobic conditions, helping to generate ATP and maintaining homeostasis (Barz et al, 2010).…”