Purification and properties of hydrogenase from Clostridium pasteurianum W5

Chen, Jiann-Shin; Mortenson, Leonard E.

doi:10.1016/0005-2795(74)90025-7

Cited by 218 publications

(71 citation statements)

References 32 publications

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“…The calculate molecular weight of the purified enzyme (56 000) is the same order of other hydrogenases characterized from bacterial sources [5,7,8] but much lower than that reported (230 000) for Anabaena cylindrica hydrogenase [ 141.…”

Section: Discussionmentioning

confidence: 63%

“…Although the enzyme activity has been found in many bacteria and algae [2], homogeneous enzyme preparations have only been isolated from photosynthetic [3-S], aerobic [6] and anaerobic bacteria [7,8]. There are a few reports about hydrogenase activity in nitrogen-fixing, heterocystous Cyanobacteria [9-121, Here we report the purification (to 1 lo-fold) and properties of hydrogenase activity from the nonheterocystous cyanobacterium S. maxima.…”

Section: Introductionmentioning

confidence: 92%

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Isolation and characterization of the hydrogenase activity from the non‐heterocystous cyanobacterium Spirulina maxima

et al. 1979

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Section: Discussionmentioning

confidence: 63%

Section: Introductionmentioning

confidence: 92%

Isolation and characterization of the hydrogenase activity from the non‐heterocystous cyanobacterium Spirulina maxima

et al. 1979

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“…F 420 was purified from Methanothermobacter thermoautotrophicus and reduced to generate F 420 H 2 employing previously described methods (59,75). C. pasteurianum ferredoxin and bidirectional hydrogenase were generous gifts from Dr. J. H. Chen of Virginia Tech (76). All other chemicals were purchased from standard suppliers.…”

Section: Methodsmentioning

confidence: 99%

“…The assays were performed in round glass cuvettes sealed with cutoff butyl rubber stoppers with N 2 (1.3 ϫ 10 5 Pa) in the headspace (27), except with ferredoxin as electron carrier H 2 (1.3 ϫ 10 5 Pa) was used. Ferredoxin was from C. pasteurianum, and it was reduced in situ with bidirectional hydrogenase from the same organism, and the respective final concentrations in the assay were 5 M and 50 nM, respectively (76).…”

Section: Methodsmentioning

confidence: 99%

A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD

Susanti¹,

Loganathan²,

Mukhopadhyay

2016

Journal of Biological Chemistry

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A recent report suggested that the thioredoxin-dependent metabolic regulation, which is widespread in all domains of life, existed in methanogenic archaea about 3.5 billion years ago. We now show that the respective electron delivery enzyme (thioredoxin reductase, TrxR), although structurally similar to flavincontaining NADPH-dependent TrxRs (NTR), lacked an NADPH-binding site and was dependent on reduced coenzyme F 420 (F 420 H 2 ), a stronger reductant with a mid-point redox potential (E 0 ) of ؊360 mV; E 0 of NAD(P)H is ؊320 mV. Because F 420 is a deazaflavin, this enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). It transferred electrons from F 420 H 2 to thioredoxin via proteinbound flavin; K m values for thioredoxin and F 420 H 2 were 6.3 and 28.6 M, respectively. The E 0 of DFTR-bound flavin was approximately ؊389 mV, making electron transfer from NAD(P)H or F 420 H 2 to flavin endergonic. However, under high partial pressures of hydrogen prevailing on early Earth and present day deep-sea volcanoes, the potential for the F 420 /F 420 H 2 pair could be as low as ؊425 mV, making DFTR efficient. The presence of DFTR exclusively in ancient methanogens and mostly in the early Earth environment of deep-sea volcanoes and DFTR's characteristics suggest that the enzyme developed on early Earth and gave rise to NTR. A phylogenetic analysis revealed six more novel-type TrxR groups and suggested that the broader flavin-containing disulfide oxidoreductase family is more diverse than previously considered. The unprecedented structural similarities between an F 420 -dependent enzyme (DFTR) and an NADPH-dependent enzyme (NTR) brought new thoughts to investigations on F 420 systems involved in microbial pathogenesis and antibiotic production.

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“…Partial purification of hydrogenase (hydrogen:ferredoxin oxidoreductase, EC 1.12.7.1) from the same microorganism followed the method of Nakos and Mortenson (18) with the omission of protamine sulfate precipitation and Sephadex column chromatography, these steps being replaced by hydroxylapatite column treatment (19). The specific activity of the final preparation was essentially that of the enzyme preparations described by Lappi et al (6).…”

Section: Methodsmentioning

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.

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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...

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Purification and properties of hydrogenase from Clostridium pasteurianum W5

Cited by 218 publications

References 32 publications

Isolation and characterization of the hydrogenase activity from the non‐heterocystous cyanobacterium Spirulina maxima

Isolation and characterization of the hydrogenase activity from the non‐heterocystous cyanobacterium Spirulina maxima

A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD

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

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