Properties of the tungsten‐substituted molybdenum formylmethanofuran dehydrogenase from <i>Methanobacterium wolfei</i>

Schmitz, Ruth A.; Albracht, S.P.J.; Thauer, Rudolf K.

doi:10.1016/0014-5793(92)80743-z

Cited by 49 publications

(59 citation statements)

References 23 publications

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“…The significance of this inhomogeneity is not known. Note that a similar inhomogeneity in the formylmethanofuran dehydrogenase from M. wolfei has been assigned to an unexpectedly large hyperfine splitting from 183 W hyperfine interaction (28). Further studies are required to elucidate the nature of these inhomogeneities.…”

Section: Discussionmentioning

confidence: 87%

“…This observation is remarkable in view of previously reported data on naturally occurring tungsten enzymes. Highly unusual g values were reported for W(V) in C. thermoaceticum formate dehydrogenase (g ϭ 2.10 [8]) and W(V) in Methanobacterium wolfei formylmethanofuran dehydrogenase (g ϭ 2.05 [28]). It is also interesting that the tungsten in P. furiosus aldehyde oxidoreductase has been reported to be of extremely low potential, the W(VI)/W(V) couple having a reduction potential of ϽϪ500 mV (11), while we find for the D. gigas aldehyde dehydrogenase that the W(V)/W(IV) couple has an E m,7.5 of ϾϪ400 mV.…”

Section: Discussionmentioning

confidence: 92%

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Purification and characterization of a benzylviologen-linked, tungsten-containing aldehyde oxidoreductase from Desulfovibrio gigas

1995

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Desulfovibrio gigas NCIMB 9332 is a sulfate-reducing bacterium which is able to grow with H 2 , L-and D-lactate, C 4 -dicarboxylic acids, and ethanol as energy sources (17,35). D. gigas not only oxidizes ethanol (to acetate) but it can also store massive amounts of polyglucose (29) which under fermentative conditions can be degraded with ethanol as a major end product (26).Recently, we characterized the NAD-dependent alcohol dehydrogenase from D. gigas and showed it to be an oxygen-labile, decameric enzyme (14). From lactate-grown cells, a molybdenum iron-sulfur protein (MOP) which had 2,6-dichlorophenol-indophenol (DCPIP)-linked aldehyde dehydrogenase activity (1, 31) was purified. In extracts of cells grown on lactate or ethanol, high levels of activity of a coenzyme A-independent, benzylviologen-linked aldehyde dehydrogenase (BV-AlDH) were measured (17). Because at that time only the MOP was known as an enzyme with aldehyde dehydrogenase activity in D. gigas, it was suggested that the BV-AlDH activity was catalyzed by the MOP. Recently, we found that the addition of 0.1 M tungstate to the medium had a strongly stimulatory effect on the growth of D. gigas on ethanol. Omission of tungstate from the medium resulted in a decrease or absence of the BV-AlDH activity and an increase of the DCPIP-linked aldehyde dehydrogenase activity (13). These data and the different characteristics of the enzyme activities in cell extracts strongly suggested that the BV-AlDH and the MOP are two different enzymes.In this report, we describe the purification and characterization of the BV-AlDH and provide evidence that this enzyme belongs to the group of molybdopterin-and tungsten-containing aldehyde:acceptor oxidoreductases. MATERIALS AND METHODSOrganism, cultivation, and preparation of cell extract. D. gigas NCIMB 9332 was cultivated on ethanol, harvested, washed, and stored as described earlier (14), with the following minor modifications. The washing buffer was 50 mM potassium phosphate (pH 7.5) containing 2 mM dithiothreitol (DTT) and 1 mM dithionite. Cell extract was prepared by three successive passages through a French pressure cell operated at 106 MPa and centrifugation (20 min at 48,000 ϫ g and 4ЊC). The cell extract was then used for enzyme purification or stored at Ϫ20ЊC under N 2 until further use.Enzyme assays. Assay systems in cuvettes were made anaerobic by bubbling with N 2 for at least 3 min and were then closed with grey butyl rubber stoppers. The standard assay contained 50 mM potassium phosphate buffer (pH 7.5) and 2 mM benzylviologen in a total volume (after the following additions) of 1 ml. Then, 5 l of a freshly prepared dithionite solution (1.5 mM) and enzyme solution (usually 10 l) were added with microsyringes and the reaction was started by adding acetaldehyde to a final concentration of 1 mM; oxygen was removed from acetaldehyde stock solutions in crimp-seal bottles by replacing the atmosphere with oxygen-free nitrogen twice. Addition of dithionite was necessary to prevent a lag phase in the reaction. Ac...

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Section: Discussionmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 92%

Purification and characterization of a benzylviologen-linked, tungsten-containing aldehyde oxidoreductase from Desulfovibrio gigas

1995

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“…However, the tungsten substituted molybdenum isoenzyme from M. wolfei exhibited after oxidation an EPR signal which was clearly derived from tungsten as indicated by characteristic hyperfine splitting (Schmitz et al, 1992b). This indicates that the redox potentials of the W(VI)/(V) couple and of the W(V)/(IV) couple in the tungsten-substituted molybdenum isoenzyme are less negative and further apart than in the tungsten isoenzymes.…”

Section: Epr Propertiesmentioning

confidence: 89%

Formylmethanofuran dehydrogenases from methanogenic Archaea Substrate specificity, EPR properties and reversible inactivation by cyanide of the molybdenum or tungsten iron‐sulfur proteins

Bertram

Karrasch

Schmitz

et al. 1994

European Journal of Biochemistry

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Formylmethanofuran dehydrogenases, which are found in methanogenic Archaea, are molybdenum or tungsten iron-sulfur proteins containing a pterin cofactor. We report here on differences in substrate specificity, EPR properties and susceptibility towards cyanide inactivation of the enzymes from Methanosarcina barkeri, Methanobacterium thermoautotrophicum and Methanobacterium woljei.The molybdenum enzyme from M. barkeri (relative activity with N-formylmethanofuran = 100%) was found to catalyze, albeit at considerably reduced apparent V,,,,,, the dehydrogenation of N-furfurylformamide (11 %), N-methylformamide (0.2%), formamide (0.1 %) and formate (1 %). The molybdenum enzyme from M. wolfei could only use N-furfurylformamide (1 %) and formate (3 %) as pseudosubstrates. The molybdenum enzyme from M. thermoautotrophicum and the tungsten enzymes from M. thermoautotrophicum and M. wolfei were specific for N-formylmethanofuran.The molybdenum formylmethanofuran dehydrogenases exhibited at 77 K two rhombic EPR signals, designated FMDred and FMD,,, both derived from Mo as shown by isotopic substitution with 9 7 M~. The FMD,, signal was only displayed by the active enzyme in the reduced form and was lost upon enzyme oxidation; the FMD,, signal was displayed by an inactive form and was not quenched by 0,. The tungsten isoenzymes were EPR silent.The molybdenum formylmethanofuran dehydrogenases were found to be inactivated by cyanide whereas the tungsten isoenzymes, under the same conditions, were not inactivated. Inactivation was associated with a characteristic change in the molybdenum-derived EPR signal. Reactivation was possible in the presence of sulfide. Abbreviations. FMD,,,, EPR signal attributed to active formylmethanofuran dehydrogenase ; FMD,,, EPR signal attributed to inactive formylmethanofuran dehydrogenase ; R, Land6 splittina + co2 t P IE" = -497 mV (Thauer, 1990) factor; gx, gy-and g,, Land6 splitting factors in the x , y and directions for anisotropic systems; 1 U = 1 Fmol formylmethanofuran dehydrogenatedmin.The physiological electron accePtor/donor is not knownMethylviologen (E"' = -446 mV) can be used as artificial

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“…The different chromatographic behaviour is probably not due to the fact that the one enzyme contains molybdenum and the other tungsten. When formylmethanofuran dehydrogenase I was isolated from cells grown on medium supplemented with molybdate plus tungstate the purified enzyme contained both transition metals without this having an cffect on the chromatographic propcrties (Schmitz et al, 1992b). I t therefore has to be postulatcd that formylmethanofuran dchydrogenases 1 and 11 differ in their primary structure and/or in another structural feature.…”

Section: Discussionmentioning

confidence: 99%

A molybdenum and a tungsten isoenzyme of formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei

Schmitz

Albracht

Thauer

1992

European Journal of Biochemistry

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We have recently reported that the thermophilic archaeon Methanohucterium woCfei contains two formylmethanofuran dehydrogenases, I and 11. E'ormylmethanofuran dehydrogenase 11, which is preferentially expressed in tungsten-grown cells, has been purified and shown to be a tungsten-ironsulfur protein. We have now purified and characterized formylmethanofuran dehydrogenase I from molybdenum-grown cells and shown that it is amolybdenum-iron-sulfur protein. The purified enzyme, with a specific activity of 27 U/mg protein, was found to be composed of three subunits of apparent molecular mass 64 kDa, 51 kDa, and 31 kDa and to contain per moll4h-kDa molecule approximately 0.23 mol molybdenum, 0.46 mol molybdopterin guanine dinucleotide, and 6.6 mol non-heme iron but no tungsten ( < 0.01 mol). The molybdenum enzyme differed from the tungsten enzyme (8 Ujmg) in that it catalyzed the oxidation of N-furfurylformamide and formate and was inactivated by cyanide. The two enzymes also differed significantly in the pH optimum, in the apparent K , for the electron acccptor, and in the chromatographic behaviour. The molybdenum enzyme and the tungsten enzyme were similar, however, in that the N-terminal amino acid sequences determined for the CI and fl subunits were identical up to residue 23, indicating that the two proteins are isoenzymes.The molybdenum enzyme, as isolated, was found to display an EPR signal derived from molybdenum as evidenced by isotope substitution.Formylmethanofuran dehydrogenase (Borner et al., 1989) catalyzes the reversible reduction of C 0 2 plus methanofuran (MFR) to N-formylmethanofuran (CHO-MFR). This reaction is the first step in methane formation from COz and H2 in all methanogenic archaea (DiMarco et al., 1990; Schworer and Thauer, 1991). C02+MFK+2[H] + CHO-MF:K+H20 E"'= -497 mVThe enzyme has been isolated and characterized from Methanosarcina burkeri and from Methanobacferium thermoaututruphicum (Borner et al., 1991). The dehydrogenase from these archaea (Woese et al., 1990) contains molybdenum, a molybdopterin dinucleotide, and iron-sulfur clusters. The molybdenum enzymes from M . huvkeri and from M . thermuautotruphicum differ in that the former, as isolated, is composed of six subunits of apparent molecular mass 65 kDa, 50 kDa, 37 kDa, 34 kDa, 29 kDa, and 17 kDa and the latter of only two subunits of apparent molecular mass 60 kDa and 45 kDa. They also differ significantly in substrate specificity: formylmethanofuran dehydrogenase from M. barker; is able to catalyze the oxidation of N-furfurylformamide, of formamide, of N-methylformamide, and of formate (unpublished data), whereas the enzyme from M . thermouututrophicum can only mediate the oxidation of formylmethanofuran (Breitung et al., 1990). We have recently found that Mcthnnobacterium wulfei, which is a thermophile growing at 65 "C, contains two active formylmethanofuran dehydrogenases 1 and 11, which can be separated by anion-exchange chromatography on Mono Q and by hydrophobic chromatography on Phenyl-Superose. Formylmethanofuran dehyd...

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Properties of the tungsten‐substituted molybdenum formylmethanofuran dehydrogenase from Methanobacterium wolfei

Cited by 49 publications

References 23 publications

Purification and characterization of a benzylviologen-linked, tungsten-containing aldehyde oxidoreductase from Desulfovibrio gigas

Purification and characterization of a benzylviologen-linked, tungsten-containing aldehyde oxidoreductase from Desulfovibrio gigas

Formylmethanofuran dehydrogenases from methanogenic Archaea Substrate specificity, EPR properties and reversible inactivation by cyanide of the molybdenum or tungsten iron‐sulfur proteins

A molybdenum and a tungsten isoenzyme of formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei

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