On the iron-sulfur cluster in hydrogenase from Clostridium pasteurianum W5.

Erbes, David L.; Burris, R. H.; Orme‐Johnson, William H.

doi:10.1073/pnas.72.12.4795

Cited by 93 publications

(38 citation statements)

References 22 publications

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“…The spectra ofthe reduced, oxidized, and oxidized-plus-CO forms of the enzyme have been published (9,12,13,28). The reduced enzyme samples used in this study all gave rise to an EPR spectrum identical to that given in figure 4 of ref.…”

Section: Resultsmentioning

confidence: 66%

Iron-sulfur clusters of hydrogenase I and hydrogenase II of Clostridium pasteurianum.

Adams

Eccleston

Howard

1989

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

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The iron and acid-labile sulfide contents and the electron paramagnetic resonance (EPR) properties of hydrogenase I (bidirectional) and hydrogenase II (uptake) of Clostridiumpasteurianum (strain W5) have been determined on the basis of quantitative amino acid analyses. The iron and acid-labile sulfide values are approximately 20 and 18 atoms per molecule of hydrogenase I and 14 and 11 atoms per molecule of hydrogenase II, respectively. These amounts are substantially greater than previously reported values, which relied on protein concentration determined by calorimetric assay. The oxidized hydrogenases exhibit unusual EPR signals that originate from a novel type of iron-sulfur center, termed the hydrogenase or H cluster, which covalently binds the inhibitor CO. This EPR signal represents approximately one unpaired electron per molecule in each enzyme with and without bound CO, which is consistent with the presence of one oxidized H cluster (S = 1/2) per enzyme molecule. The two enzymes also contain ferredoxin-type four-iron centers or F clusters. The EPR signals from the F clusters observed in the reduced forms ofhydrogenase I and hydrogenase II account for approximately four and one unpaired electron per molecule, respectively. We conclude from the iron determinations and the EPR results, together with a reevaluation of previous spectroscopic data, that in both hydrogenases the H cluster probably comprises six iron atoms. Mechanistic models of the two hydrogenases are presented that account for their cluster compositions and the dramatic differences in their catalytic activities.Hydrogenases catalyze the reversible activation of molecular H2 according to the reaction:They are widely distributed in bacteria and are a very heterogeneous group of enzymes: their only common feature is that they are iron-sulfur proteins and the majority also contain nickel (1-4). The anaerobic N2-fixing bacterium Clostridium pasteurianum is so far unique in that it contains two hydrogenases that lack nickel. Hydrogenase I (bidirectional) catalyzes both H2 evolution and H2 oxidation at extremely high rates in vitro, whereas hydrogenase II preferentially catalyzes H2 oxidation (5)(6)(7)(8) (7,(9)(10)(11)(12). A combination of EPR and 1H, 13C, and 57Fe electron nuclear double resonance spectroscopy (ENDOR) has shown that in both hydrogenases the H cluster contains two types of iron atoms and covalently binds CO, a potent inhibitor, without cluster destruction (13,14). Subsequent Mossbauer analysis of hydrogenase II showed that its H cluster contains two types of iron atoms in a 2:1 ratio and was '40% of the total iron (15). Therefore, it was concluded that the H cluster is a three-iron cluster (15). Thus, on the basis of these results and the determined iron concentrations, hydrogenase II was thought to contain 7 iron atoms per molecule (one H and one F cluster), and hydrogenase I, 11 iron atoms per molecule (one H and two F clusters). However, the proposed cluster compositions of the clostridial hydrogenases are not consisten...

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Section: Resultsmentioning

confidence: 66%

Iron-sulfur clusters of hydrogenase I and hydrogenase II of Clostridium pasteurianum.

Adams

Eccleston

Howard

1989

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

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“…On careful oxidation the [Fe]-hydrogenases give rise to an EPR signal with principal g-factors 2.10, 2.04 and 2.001 [2,30]. This signal has been assigned to the 'H-clusters'.…”

Section: Discussionmentioning

confidence: 99%

Evidence for an [Fe]‐type hydrogenase in the parasitic protozoan Trichomonas vaginalis

1993

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The hydrogenase of the pathogenic protozoan Trzchomonas vugmalis was extracted and partially purified. The catalytic and spectroscopic properties of the enzyme indicate that it belongs to the class of [Fe]-hydrogenases. rather than the [NiFe]-hydrogenases. The hydrogenase activity was highly sensitive to carbon monoxide, 50% inhibition being attained by 1 PM CO. The EPR spectrum of the most active fractions from chromatography, after reduction by hydrogen and partial reoxidation under argon, showed an EPR spectrum at g = 2.10, 2.04, 2.00. This unusual spectrum is characteristic of the 'H-cluster', as seen in [Fe]-hydrogenases of anaerobic bacteria such as Clostrldium spp.

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“…Such enzyme forms might be a series of different oxidation states as is known to exist for the C. pasteurianum hydrogenase (4). That H2 antagonizes the protective effect of CO against 02 inactivation of hydrogenase and is competitive in CO inhibition may support a hypothesis that CO and 02 bind to the active site.…”

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confidence: 81%

Inactivation of Hydrogenase in Cell-free Extracts and Whole Cells of Chlamydomonas reinhardi by Oxygen

Erbes¹,

King²,

Gibbs³

1979

Plant Physiol.

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02 irreversibly inactivates hydrogenase from Chlamydomonas reinhardiThe mechanism for the inactivation involves the reaction of one molecule of hydrogenase with one molecule of 02 (or two oxygen atoms) in the transition complex of the rate-limiting step. The second order rate constant for this reaction is 190 atmospheres-' minute-1 (1.4 x 105 molar-' minute-1). At levels above 0.01 atmosphere 02, the increased numbers of 02 molecules may compete for the site of inactivation hindering the proper orientation for inactivation of any one 02 molecule and resulting in lowered rates of inactivation.CO is a reversible inhibitor of hydrogenase acting competitively against H2. The Ki for CO is 0.0010 atmosphere. CO antagonizes 02 inactivation. In a period when complete inactivation by 02 would usualHy occur, the presence of CO greatly reduces the inactivation rate.After 3 hours of adaptation in whole cells, the presence of H2 lowers the rate of deadaptation of hydrogenase. Inasmuch as H2 promotes increased 02 uptake the cellular concentration of 02 is likely to be lower. After 48 hours of adaptation 02 uptake is reduced even when H2 is present and the pattern of deadaptation under 02 with and without H2 and CO is qualitatively the same as observed for the inactivation of cell-free hydrogenase. The mechanism of inactivation of celi-free hydrogenase by 02 may be the same as the mechanism for loss of hydrogenase during deadaptation in whole algal cells.Since the initial observation of H2 metabolism in green algae the sensitivity of algal H2 metabolism to O2 has been noted (5). Simultaneous uptake of H2 and O2 (the oxyhydrogen reaction) is inhibited when the O2 level exceeds a specific concentration (6, 10). Also, the photoproduction of 02 in algae will cause cessation of H2 metabolism. Removal of photoproduced O2 reversed the inhibition of hydrogenase (9) C. moewusii was grown in minimal medium plus acetate (7).The hydrogenase was prepared as above.Assays. Protein was assayed by the biuret method (8) and Chl was assayed spectrophotometrically in 80% acetone (2). The enzyme was assayed by MV3 reduction followed on a Gilford recording spectrophotometer at 605 nm. Hydrogenase solution was injected into a cuvette with 2.0 ml of anaerobically prepared 10 mM MV in 50 mm Tris-HCl (pH 8.0) under 1.0 atm H2 at 25 C. The extinction coefficient for reduced MV was determined to be 9.6 mM-' cm-' by reduction with excess sodium dithionite at pH 8.0. One unit of hydrogenase activity is defined as the reduction of 1.0 mol MV/min.

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On the iron-sulfur cluster in hydrogenase from Clostridium pasteurianum W5.

Cited by 93 publications

References 22 publications

Iron-sulfur clusters of hydrogenase I and hydrogenase II of Clostridium pasteurianum.

Iron-sulfur clusters of hydrogenase I and hydrogenase II of Clostridium pasteurianum.

Evidence for an [Fe]‐type hydrogenase in the parasitic protozoan Trichomonas vaginalis

Inactivation of Hydrogenase in Cell-free Extracts and Whole Cells of Chlamydomonas reinhardi by Oxygen

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