Cell-free Hydrogenase from Chlamydomonas

Abeles, Fredrick B.

doi:10.1104/pp.39.2.169

Cited by 52 publications

(18 citation statements)

References 23 publications

(10 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Several organic substrates stimulate the photoevolution of H2 in Ankistrodesmus braunii (19). Finally, cell-free preparations of Chlamydomonas eugametos evolve H2 from reduced pyridine nucleotides (1). These observations suggest that some algae may be able to obtain reductant for the photoevolution of H2 from the oxidation of organic compounds as well as from the photooxidation of water.…”

mentioning

confidence: 86%

The Mechanism of Hydrogen Evolution by Chlamydomonas moewusii

Healey

1970

Plant Physiol.

View full text Add to dashboard Cite

Using manometric techniques, H2 evolution in both darkness and light has been studied in the green alga, Chlamydomonas moewusii.Hydrogen evolution in the dark is accompanied by the release of only CO2 in manometrically detectable amounts. It is depressed by dark starvation and inhibited both by monofluoroacetic acid and by uncouplers of phosphorylation. This evidence suggests that the reaction is dependent on oxidative carbon metabolism for reductant and phosphorylation for energy to raise the reductant to a redox potential capable of reducing H+.Photoevolution of H2 is also accompanied by the release of only CO2. It is depressed by dark starvation and stimulated by acetate or a period of photosynthesis. Monofluoroacetic acid causes complete inhibition, while 3-(3,4-dichlorophenyl)-1,1-dimethylurea causes no or only slight inhibition. These results indicate that oxidative carbon metabolism is the source of reductant for the reaction. Photoevolution of H2 does not show Emerson enhancement, and it has an action spectrum peaking at a longer wave length than that of photosynthesis. These characteristics, together with the slight effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea on the reaction, show that only system I of photosynthetic electron transport is involved in the reaction. Photoevolution of H2 is stimulated by uncouplers; this indicates that the role of light is not to provide energy by phosphorylation. Rather, the results support an electron flow driven directly by light through system I from reductant produced in oxidative carbon metabolism to a redox potential capable of reducing H+.reactions were involved in the dark and light. They suggested that the dark reaction depended on the oxidation of organic compounds for reductant, while that in the light derived reductant from the photoxidation of water. The former suggestion has been supported by the stimulatory effects both of glucose and of periods of photosynthesis of H2 release in the dark by S. obliquus (11) and the dependence of H2 release by Chlorella pyrenoidosa on an exogenous substrate (8). The dependence of photoevolution of H2 on photooxidation of water has been supported by the observation of the simultaneous release of H2 and O2 in the light by anaerobic Chlorella sp. (25,26). Further support has come from the observation by Bishop and Gaffron (4) of several correlations between the release of H2 and 02 by S. obliquus. In this alga, the simultaneous release of H2 and 02 in the approximate molar ratio of 2:1 was observed. Both the photoevolution of H2 and that of 02 were inhibited to the same extent by mutations, inhibitors of the Hill reaction, and manganese deficiency. Finally, both showed similar enhancements in combined red and far-red light. Neither MFA nor iodoacetic acid had any effect on the photoevolution of H2 in this species.The above results suggest that only electron transport from water through the two photosystems of photosynthesis to the release of H2 is involved in the photoevolution of H2 by algae. However, a few obse...

show abstract

mentioning

confidence: 86%

The Mechanism of Hydrogen Evolution by Chlamydomonas moewusii

Healey

1970

Plant Physiol.

View full text Add to dashboard Cite

show abstract

“…In addition to binding CO and CN, each of the iron atom coordinates a bridging organic ligand thought to be a di(thio-methyl)amine moiety [12]. An important limitation for this biohydrogen photoproduction is that hydrogenase only operates under anaerobic conditions because its expression, activation and activity is sensitive to oxygen [13][14][15][16][17]. In microalgae, the enzyme is quickly inactivated by photosystem (PS) II-dependent O 2 evolution upon illumination.…”

Section: Introductionmentioning

confidence: 99%

A novel screening method for hydrogenase-deficient mutants in Chlamydomonas reinhardtii based on in vivo chlorophyll fluorescence and photosystem II quantum yield

Godaux

Emonds-Alt

Berne

et al. 2013

International Journal of Hydrogen Energy

View full text Add to dashboard Cite

In Chlamydomonas reinhardtii, prolonged anaerobiosis leads to the expression of enzymes belonging to various fermentative pathways. Among them, oxygen-sensitive hydrogenases (HydA1/2) catalyze the synthesis of molecular hydrogen from protons and reduced ferredoxin in the stroma. In this work, by analyzing wild type and mutants affected in H2 production, we show that maximal PSII photosynthetic electron transfer during the first seconds of illumination after a prolonged darkanaerobiosis period is linearly related to hydrogenase capacity. Based on the specific chlorophyll fluorescence induction kinetics typical of hydrogenase-deficient mutants, we set up an in vivo fluorescence imaging screening protocol allowing to isolate mutants impaired in hydrogenase expression or activity, as well as mutants altered in related metabolic pathways required for energy production in anaerobiosis. Compared to previously described screens for mutants impaired in H2 production, our screening method is remarkably fast, sensitive and non invasive. Out of 3000 clones from a small-sized insertional mutant library, five mutants were isolated and the most affected one was analyzed and shown to be defective for the hydrogenase HydG assembly factor.Pierre Cardol, PhD, FNRS research associate Génétique des microorganismes -Institut de Botanique B22 Boulevard du Rectorat, 27 -Université de Liège-B-4000 Liège, Belgique Tel/Fax +32 4 3663840 e-mail: Pierre.cardol@ulg.ac.be Liège, 07 november 2012 Dear Editor, Please find here enclosed a revised version of our manuscript entitled 'A novel screening method for hydrogenase-deficient mutants in Chlamydomonas reinhardtii based on in vivo chlorophyll fluorescence and photosystem II quantum yield.' that had received a positive feedback from the first round of reviewing.We are grateful to you for having encouraged us to resubmit quickly a modified manuscript. We have addressed all the comments raised by the reviewers on our first submission. A point to point answer has been submitted along with the MS. Main changes are : (i) the addition of a new experimental set of data (hydrogen evolution measurements in all mutant strain and some control experiments) and (ii) all data that were previously not shown are now included in the manuscript in main tables or as supplemental figures.We hope that we have now improved our manuscript so you'll find it suitable for publication in International Journal of Hydrogen Energy. Sincerely yours,On behalf all coauthors, Pierre Cardol *Cover LetterReviewer #1:Major points:I. Please measure and show, respectively, the hydrogenase activity of the wild types or control strains (like respiratory mutants, pfl1-mutant) in your setup. Available literature shows that absolute values can differ significantly depending on exact experimental setup and strain. As the correlation between PSII quantum yield and hydrogenase activity is the major point of your study, this data is essential, and citing hydrogenase activities reported in literature is not sufficient.Answer : the...

show abstract

“…Efforts to achieve stabilization to date (6-10) have been empirical, involving mainly immobilization of the enzyme (6, 9, 10), but success has been limited. It is known (1,11) that the rate of 02 inactivation in solutions of the enzyme is directly proportional to 02 concentration. We have conceived a rationale based on exploitation of the well-known "salting out" phenomenon whereby the concentration of dissolved gases in aqueous solution is reduced as the salt concentration is increased (12).…”

mentioning

confidence: 99%

A rationale for stabilization of oxygen-labile enzymes: application to a clostridial hydrogenase.

Klibanov¹,

Kaplan²,

Kamen³

1978

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

ABSTRACr A general procedure for stabilization of 02-labile enzymes exploiting "salting out" of oxygen from the microenvironment in the molecular layers immediately adjacent to charged surfaces of polyionic solid adsorbents has been develope. Empirical verification of this rationale is provided. The half-life of air inactivation of the Og-labile hydrogenase (EC 1.12.7.1) from Clostridium pasteurianum is increased 20-to 2-fold simply by adsorption (noncovalent binding) in dilute Tris-HCI buffer on common anion exchange supports such as DEAE-cellulose or Dowex 1-X2. Predicted increases in degree of stabilization by using more densely charged adsorbents (such as polyethyleneimine-cellulose), as well as bulkier solvent counter-anions, are found; half-lives for air inactivation for the bound hydrogenase can be increased to 3000-fold longer than that of the free enzyme. Most of the total catalytic activity, assayed as H2 evolution from dithionite mediated by methyl viologen or ferredoxin, is retained, whereas the expected suppression of H2 uptake in the reverse reaction is observed. Stabilization of the oxygen-labile hydrogenase (1) proposed for use in various schemes for bioconversion of solar energy (2) is critical for practical applications (3-5). Efforts to achieve stabilization to date (6-10) have been empirical, involving mainly immobilization of the enzyme (6, 9, 10), but success has been limited. It is known (1, 11) that the rate of 02 inactivation in solutions of the enzyme is directly proportional to 02 concentration. We have conceived a rationale based on exploitation of the well-known "salting out" phenomenon whereby the concentration of dissolved gases in aqueous solution is reduced as the salt concentration is increased (12). This effect is thought to be caused by competition between salt and gas for solvent, the salt component progressively removing water by hydration, thereby decreasing solvent volume available to the gas. Thus, a decrease of an order of magnitude in gas solubility occurs as solvent is saturated by salt (13,14). We are able to show that relatively simple procedures based on this fact can be developed to achieve increases in hydrogenase stability in air sufficient to meet the requirements for practical applications in solar energy bioconversion.Our rationale is that if hydrogenase, or any Orlabile enzyme, is attached to a polyionic surface (it must be polycationic for an acidic enzyme such as hydrogenase to be adsorbed), the enzyme molecules will be buried in a microenvironment equivalent to that provided by an extremely concentrated salt solution. There will be little solvent available for gas solution, so that access to the adsorbed enzyme will be minimal. In all probability, the salting out of gas may be much greater than is possible with saturated salt solutions because of solubility limitations. Hence the 02 concentration in such a microenvironment, extending several molecular layers from the charged adsorbent surface, will be very small so that a relatively large increase in 02 st...

show abstract

Cell-free Hydrogenase from Chlamydomonas

Cited by 52 publications

References 23 publications

The Mechanism of Hydrogen Evolution by Chlamydomonas moewusii

The Mechanism of Hydrogen Evolution by Chlamydomonas moewusii

A novel screening method for hydrogenase-deficient mutants in Chlamydomonas reinhardtii based on in vivo chlorophyll fluorescence and photosystem II quantum yield

A rationale for stabilization of oxygen-labile enzymes: application to a clostridial hydrogenase.

Contact Info

Product

Resources

About