Continuous monitoring of the activation and activity of 6NiFe9-hydrogenases by membrane-inlet mass spectrometry

Vignais, Paulette M.; Cournac, Laurent; Hatchikian, E.C.; Elsen, Sylvie; Serebryakova, Larissa T.; Зорин, Н. А.; Dimon, Bernard

doi:10.1016/s0360-3199(02)00114-3

Cited by 41 publications

(48 citation statements)

References 42 publications

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“…Several differences can be noted concerning the regulatory hydrogenases. Whereas both HupUV (47) and HoxBC (3) are able to produce H 2 in the presence of D 2 during H-D exchange reaction experiments, HD formation was detected only with HupUV, as previously demonstrated (45) and recently confirmed (A. L. De Lacey, personal communication). Therefore, there are differences at the molecular and mechanistic levels between these two proteins.…”

Section: Discussionsupporting

confidence: 76%

“…This promoter is very active, since in R. capsulatus grown under optimal conditions, the nitrogenase proteins could account for up to 20% of the total cell extract (22). To facilitate purification of HupUV, a His 6 tag was inserted at the N terminus of HupU, and the presence of this tag did not impair hydrogenase activity of the protein, as previously reported (45). Thus, a one-step purification on an Ni 2ϩ -charged column was sufficient to obtain about 90% pure HupUV.…”

Section: Discussionmentioning

confidence: 87%

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Interaction between the H ₂ Sensor HupUV and the Histidine Kinase HupT Controls HupSL HydrogenaseSynthesis in Rhodobactercapsulatus

2003

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The photosynthetic bacterium Rhodobacter capsulatus contains two [NiFe]hydrogenases: an energy-generating hydrogenase, HupSL, and a regulatory hydrogenase, HupUV. The synthesis of HupSL is specifically activated by H 2 through a signal transduction cascade comprising three proteins: the H 2 -sensing HupUV protein, the histidine kinase HupT, and the transcriptional regulator HupR. Whereas a phosphotransfer between HupT and HupR was previously demonstrated, interaction between HupUV and HupT was only hypothesized based on in vivo analyses of mutant phenotypes. To visualize the in vitro interaction between HupUV and HupT proteins, a six-His (His 6 )-HupU fusion protein and the HupV protein were coproduced by using a homologous expression system. The two proteins copurified as a His 6 -HupUHupV complex present in dimeric and tetrameric forms, both of which had H 2 uptake activity. We demonstrated that HupT and HupUV interact and form stable complexes that could be separated on a native gel. Interaction was also monitored with surface plasmon resonance technology and was shown to be insensitive to salt concentration and pH changes, suggesting that the interactions involve hydrophobic residues. As expected, H 2 affects the interaction between HupUV and HupT, leading to a weakening of the interaction, which is independent of the phosphate status of HupT. Several forms of HupT were tested for their ability to interact with HupUV and to complement hupT mutants. Strong interaction with HupUV was obtained with the isolated PAS domain of HupT and with inactive HupT mutated in the phosphorylable histidine residue, but only the wild-type HupT protein was able to restore normal H 2 regulation.

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Section: Discussionsupporting

confidence: 76%

Section: Discussionmentioning

confidence: 87%

Interaction between the H ₂ Sensor HupUV and the Histidine Kinase HupT Controls HupSL HydrogenaseSynthesis in Rhodobactercapsulatus

2003

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“…Thus, despite sequence homologies between the Synechocystis and R. eutropha enzymes, their characteristics are not identical. Another example of a [NiFe]hydrogenase insensitive to O 2 is the H 2 sensor HupUV of Rhodobacter capsulatus, which is able to catalyze H-D exchange in the presence of high levels of O 2 (43). In both cases, O 2 insensitivity has been assumed to result from steric hindrance preventing O 2 access to Ni.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2004

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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...

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“…This paper addresses the development of a multi-membrane inlet front end and an incorporated method of improving analytical signal using near infrared (NIR) laser diodes as a means of increasing sample permeability and desorption from the membrane in a portable system. This usage of a selectively permeable membrane is especially advantageous and has been used in other projects because it allows for an almost immediate analysis with little sample preparation [6][7][8][9]. Owing to the pressure conditions of mass spectrometry being so extreme, it is difficult to design a membrane-preferably planar-that can withstand the difference in pressure.…”

Section: Introductionmentioning

confidence: 99%

Development of Multi-Membrane Near-Infrared Diode Mass Spectrometer for Field Analysis of Aromatic Hydrocarbons

Mach

Wright

Verbeck

2014

J. Am. Soc. Mass Spectrom.

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Abstract. Membrane Inlet Mass Spectrometry (MIMS) is a technique that incorporates a semi-permeable membrane selective for differing organic molecules and chemistries. This eliminates the need for time-consuming sample preparation and facilitates near instantaneous analysis. This study will examine how the front end of MIMS incorporates three dual inlet ports, allowing for differing MIMS materials and selectivity for specific environments. Polydimethylsiloxane (PDMS) membranes have proven to be selective of benzene, toluene, and xylene (BTX) as well as aromatic hydrocarbons that are common in petroleum products while remaining selective against the aliphatic chains. PDMS has proven to be a successful choice of membrane with high permeability in atmospheric environments. In addition, polycyclic aromatic hydrocarbons (PAHs) such as acenaphthene, acenapthylene, naphthalene, and fluorene have recently been detected to the 5 ppb level in a nitrogen atmosphere with our current configuration. This preliminary work provides proof of concept using near-infrared laser diodes that act upon the membrane to increase its permeability and provide higher sensitivity of aromatic samples.

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Continuous monitoring of the activation and activity of 6NiFe9-hydrogenases by membrane-inlet mass spectrometry

Cited by 41 publications

References 42 publications

Interaction between the H ₂ Sensor HupUV and the Histidine Kinase HupT Controls HupSL HydrogenaseSynthesis in Rhodobactercapsulatus

Interaction between the H ₂ Sensor HupUV and the Histidine Kinase HupT Controls HupSL HydrogenaseSynthesis in Rhodobactercapsulatus

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

Development of Multi-Membrane Near-Infrared Diode Mass Spectrometer for Field Analysis of Aromatic Hydrocarbons

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Continuous monitoring of the activation and activity of 6NiFe9-hydrogenases by membrane-inlet mass spectrometry

Cited by 41 publications

References 42 publications

Interaction between the H 2 Sensor HupUV and the Histidine Kinase HupT Controls HupSL HydrogenaseSynthesis in Rhodobactercapsulatus

Interaction between the H 2 Sensor HupUV and the Histidine Kinase HupT Controls HupSL HydrogenaseSynthesis in Rhodobactercapsulatus

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

Development of Multi-Membrane Near-Infrared Diode Mass Spectrometer for Field Analysis of Aromatic Hydrocarbons

Contact Info

Product

Resources

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Interaction between the H ₂ Sensor HupUV and the Histidine Kinase HupT Controls HupSL HydrogenaseSynthesis in Rhodobactercapsulatus

Interaction between the H ₂ Sensor HupUV and the Histidine Kinase HupT Controls HupSL HydrogenaseSynthesis in Rhodobactercapsulatus