Catalysis of the H<sub>2</sub>-HTO Exchange by Hydrogenase. A New Assay for Hydrogenase<sup>*</sup>

Anand, Satyesh Raj; Krasna, Alvin I.

doi:10.1021/bi00888a027

Cited by 28 publications

(14 citation statements)

References 15 publications

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“…Alternatively, compound(s) present in cells during anaerobic, dark growth with CO might in some manner function as a substrate during hydrogen formation. In this event, tritium gas production could occur by an exchange reaction (33).…”

Section: Methodsmentioning

confidence: 99%

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Anaerobic growth of a Rhodopseudomonas species in the dark with carbon monoxide as sole carbon and energy substrate.

Uffen¹

1976

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

210

113

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A species of Rhodopseudomonas that grows under strict anaerobic conditions in the dark and requires CO was isolated from lake and pond sediments. Although anaerobic growth in the dark occurs in a chemically defined mineral medium with CO as the only carbon and energy source, growth is stimulated by adding trypticase. Under these conditions, cells exhibit a generation time of 6.7 hr and reach a final concentration of I to 3 X 109 cells per ml of liquid medium. Resting suspensions of CO-grown cells metabolize about 6.7 Vmol of CO per mg of protein in 1 hr and produce equimolar amounts of CO2 and H2 according to the equation CO + H210 -CO2 + H2. As predicted by this equation, when cells were suspended in tritium-labeled water containing potassium phosp ate buffer at pH 7.0 and incubated with pure CO, 3H2 gas was produced at linear rate with a constant specific activity. Carbon monoxide is a natural by-product in a variety of biologic and chemical reactions that occur in nature (1-5). Once formed, the gas may be oxidized to carbon dioxide by photochemical reactions in the troposphere (6). Evidence that biologic reactions also occur for oxidation and/or utilization of CO has been more difficult to obtain (7,8). Both plant and bacterial systems have been implicated.The ability of leaf tissue from young bean plants to oxidize CO to CO2 in light led to speculation that plants play an important role in removing CO from the atmosphere. Nevertheless, since the reaction was not observed when other tissfies were used (2), the ability to oxidize CO may not be a general property of green plants. In addition, it has been reported that a variety of aerobic and anaerobic bacteria metabolize carbon monoxide. However, in these studies performed with suspensions of whole cejls (9) or protein extracts (10-13), the reaction(s) occurred slowly and amounts of CO oxidized to CO9 were small. Likewise a few studies (14,15) suggested certain aerobic microorganisms could grow with CO. Unfortunately, important experimental details were lacking and it was not possible to determine if the cells grew methylotrophically (7, 8), i.e., with CO as a source of carbon and energy, or if the gas was cometabolized (10, 11) with other substrates required for cell growth. It has been generally accepted that two species of methanogenic bacteria can oxidize CO to CO2 under anaerobic conditions, and with H2 can form methane gas and grow (16)(17)(18) monoxide-utilizing microorganisms were selectively isolated from anaerobic enrichment cultures. About 50 ml of reduced liquid medium (described below) was prepared, but without adding trypticase, and inoculated with a 1 g sample of anaerobic, black sediment from Crystal Lake, Urbana, Ill. or from a duck pond located outside Champaign, Ill. Enrichment cultures were incubated at 340 under a stream of sterile, oxygenfree H2-CO gas (80:20 mixture). When turbid growth developed, a sample of cells was obtained from the enrichment culture by means of a platinum inoculation loop and transferred into 50 ml of ster...

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Section: Methodsmentioning

confidence: 99%

“…Development of a cell-free system to study the CO-oxidation reaction and direct measurement of hydrogenase activity using radioisotope exchange techniques (33) …”

Section: Methodsmentioning

confidence: 99%

Anaerobic growth of a Rhodopseudomonas species in the dark with carbon monoxide as sole carbon and energy substrate.

Uffen¹

1976

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

210

113

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“…In both cases, uptake by soil appears to be dependent on biological activity (7,23). Laboratory studies with whole cells or cell-free preparations have shown that the enzyme hydrogenase will catalyze the splitting of H2 or T2-HT (1,3,19). Under proper laboratory conditions, a variety of microorganisms, including many soil isolates, will produce hydrogenase (1,5,9,12,18,28,30,32).…”

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confidence: 99%

Influences of pH, Temperature, and Moisture on Gaseous Tritium Uptake in Surface Soils

Fallon

1982

Appl Environ Microbiol

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In South Carolina surface soils, the uptake of gaseous tritium (T 2 , HT, or both) showed a broad optimal temperature response from about 20 to 50°C, with the highest rates at 35 to 45°C. The optimal pH was in the range of 4 to 7. Uptake rates declined at the wet and dry extremes in soil moisture content. Inhibition seen upon the addition of hydrogen or carbon monoxide to the soil atmosphere suggested that hydrogenase may be responsible for T 2 -HT uptake in soil. During the period of most rapid recovery in a 36-day incubation of reinoculated, sterilized soil, T 2 -HT uptake rates doubled every 2 to 4 days. Thus, T 2 -HT uptake appears to be biologically mediated. Soil uptake of T 2 -HT was not severely limited by pH, temperature, or moisture in the soils tested. Thus, rapid exchange of gaseous tritium into soil water must be expected and accounted for in modeling the isotope distributions around nuclear facilities.

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“…Although the enzymatic activity of hydrogenase is determined routinely, a number of contradictory results have been published. Despite the many features that have been described in the hydrogenase reaction, the activity of this class of enzymes has not yet been thoroughly explained (1)(2)(3)(4)(5)(6)(7)(8)(9). However, there is a consensus in the hydrogenase literature that the reaction is linear; no feedback from any parts of the reaction occurs, except that some authors admit that there might be a nonlinear feedback during the activation of the enzyme (9)(10)(11).…”

Section: Introductionmentioning

confidence: 99%

“…4. The function describing the dependence is a hyperbolic function with the power ÿ0.91(3). (Inset) The same curve in log-log representation.…”

mentioning

confidence: 99%

Theoretical Calculations on Hydrogenase Kinetics: Explanation of the Lag Phase and the Enzyme Concentration Dependence of the Activity of Hydrogenase Uptake

Ősz

Bodó

Branca

et al. 2005

Biophysical Journal

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Two models of the hydrogenase reaction cycle were investigated by means of theoretical calculations and model simulations. The first model is the widely accepted triangular hydrogenase reaction cycle with minor modifications; the second is a modified triangular model, where we have introduced an autocatalytic step into the reaction cycle. Both models include a one-step activation reaction. The theoretical calculations and model simulations corroborate the assumed autocatalytic reaction step concluded from the experimental characteristics of the hydrogenase reaction.

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Catalysis of the H₂-HTO Exchange by Hydrogenase. A New Assay for Hydrogenase^*

Cited by 28 publications

References 15 publications

Anaerobic growth of a Rhodopseudomonas species in the dark with carbon monoxide as sole carbon and energy substrate.

Anaerobic growth of a Rhodopseudomonas species in the dark with carbon monoxide as sole carbon and energy substrate.

Influences of pH, Temperature, and Moisture on Gaseous Tritium Uptake in Surface Soils

Theoretical Calculations on Hydrogenase Kinetics: Explanation of the Lag Phase and the Enzyme Concentration Dependence of the Activity of Hydrogenase Uptake

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Catalysis of the H2-HTO Exchange by Hydrogenase. A New Assay for Hydrogenase*

Cited by 28 publications

References 15 publications

Anaerobic growth of a Rhodopseudomonas species in the dark with carbon monoxide as sole carbon and energy substrate.

Anaerobic growth of a Rhodopseudomonas species in the dark with carbon monoxide as sole carbon and energy substrate.

Influences of pH, Temperature, and Moisture on Gaseous Tritium Uptake in Surface Soils

Theoretical Calculations on Hydrogenase Kinetics: Explanation of the Lag Phase and the Enzyme Concentration Dependence of the Activity of Hydrogenase Uptake

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Catalysis of the H₂-HTO Exchange by Hydrogenase. A New Assay for Hydrogenase^*