The coenzyme F420-dependent formate dehydrogenase from Methanobacterium formicicum was purified to electrophoretic homogeneity by anoxic procedures which included the addition o(f azide, flavin adenine dinucleotide (FAD), glycerol, and 2-mercaptoethanol to all buffer solutions to stabilize iictivity. The enzyme contains, in approximate miolar ratios, 1 FAD molecule and 1 molybdenum, 2 zinc, 21 to 24 iron, and 25 to 29 inorganic sulfur atoms. Denaturation of the enzyme released a molybdopterin cofactor. The enzyme has a molecular weight of 177,000 and consists of one each of two different subunits, giving the composition olol.The molecular weight of the a-subunit is 85,000, and that of the ,-subunit is 53,000. The UV-visible spectrum is typical of nonheme iron-sulfur flavoprotein. Reduction of the enzyme facilitated dissociation of FAD, and the FAD-depleted enzyme was unable to reduce coenzyme F420. Preincubation of the FAD-depleted enzyme with FAD restored coenzyme F420-dependent activity.The methanogenic bacteria are phylogenetically distant from eubacteria and eucaryotes (10). Consistent with this division, they utilize several unusual cofactors, including the low-potential electron carrier coenzyme F420 (8-hydroxy-5-deazaflavin) (6). Several oxidation and reduction reactions are linked to coenzyme F420, including the oxidation of formate catalyzed by formate dehydrogenase, which supplies electrons for the reduction of carbon dioxide to methane (8,16,27,30).Spectroscopic studies of the formate dehydrogenase from Methanobacterium formicicum indicate the presence of molybdenum and iron-sulfur centers (1). Present methods for the purification of this enzyme result in a decrease in the rate of coenzyme F420 reduction relative to methyl viologen reduction (27, 28), but preincubation with flavin adenine dihucleotide (FAD) restores coenzyme F420-dependent activity, sUggesting that FAD is an essential component (28). Studies have also identified a fluorescent pterin compound associated with the enzyme (27; H. D. May, N. L. Schauer, and J. G. Ferry, submitted for publication). The quantitation of components in coenzyme F420-dependent enzymes is necessary to further understand their function in intramolecular electron transfer. Here we report on the composition and physical properties of the formate dehydrogenase from M. formicicum purified to electrophoretic homogeneity by methods that preserve coenzyme F420-dependent activity.MATERIALS AND METHODS Cell material. M. formicicum JF1 (DSM 2639) was grown in 12-liter batches as described previously (26), except that the basal medium contained the following constituents (grams per liter): NaHCO2, 6.0; NH4Cl, 1.48; K2HPO4, 1.36; KH2PO4, 0.90; NaCl, 0.45; MgSO4, 0.045; CaCl2 * 2H20, 0.06; NaCH3CO2, 2.0; Na2CO3, 3.0; Na2MoO4, 0.024; cysteine hydrochloride, 0.27; Na2SeO3, 0.0002; Na2S -9H20, 0.27; Fe(NH4)(SO4)2, 0.06; and resazurin, 0.001. Cultures were sparged with H2-CO2 (4:1) at 300 ml/min. * Corresponding author. t Present address: Department of Microbiology, University of ...

Methanobacterium formicicum strain JF-1 was cultured with formate as the sole energy source in a pH-stat fermentor. Growth was exponential, and both methane production and formate consumption were linear functions of the growth rate. Hydrogen was produced in only trace amounts, and the dissolved H 2 concentration of the culture medium was below 1 μM. The effect of temperature or pH on the rate of methane formation was studied with a single fermentor culture in mid-log phase that was grown with formate under standard conditions at 37°C and pH 7.6. Methane formation from formate occurred over the pH range from 6.5 to 8.6, with a maximum at pH 8.0. The maximum temperature of methanogenesis was 56°C. H 2 production increased at higher temperatures. Hydrogen and formate were consumed throughout growth when both were present in saturating concentrations. The molar growth yields were 1.2 ± 0.06 g (dry weight) per mol of formate and 4.8 ± 0.24 g (dry weight) per mol of methane. Characteristics were compared for cultures grown with either formate or H 2 -CO 2 as the sole energy source at 37°C and pH 7.6; the molar growth yield for methane of formate cultures was 4.8 g (dry weight) per mol, and that of H 2 -CO 2 cultures was 3.5 g (dry weight) per mol. Both formate and H 2 -CO 2 cultures had low efficiencies of electron transport phosphorylation; formate-cultured cells had greater specific activities of coenzyme F 420 than did H 2 -CO 2 -grown cultures. Hydrogenase, formate dehydrogenase, chromophoric factor F 342 , and low levels of formyltetrahydrofolate synthetase were present in cells cultured with either substrate. Methyl viologen-dependent formate dehydrogenase was found in the soluble fraction from broken cells.

Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50 ,umol of methyl viologen per min per mg of protein and 8.2 ,umol of coenzyme F420 per min per mg of protein. The apparent Km for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F420, was 10-fold greater (63 FLM) than for coenzyme F420 (6 FM). The purified enzyme also reduced flavin mononucleotide (Km = 13 ,uM) and flavin adenine dinucleotide (Km = 25 ,uM) with formate, but did not reduce NAD+ or NADP+. The reduction of NADP+ with formate required formate dehydrogenase, coenzyme F420, and coenzyme F420:NADP+ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F420. The optimal reaction rate occurred at 55°C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 FM), azide (Ki = 39 ,uM), a,a-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm. Formate dehydrogenase (FDH) is present in the aerobic, the anaerobic, and facultatively anaerobic eubacteria. The FDHs present in the anaerobes Clostridium acidiurici (27), C. formicoaceticum (21), and C. thermoaceticum (1) are 02-sensitive, require selenium and either molybdenum or tungsten for activity, and are inhibited by cyanide. The highly purified, 02-sensitive FDH from Vibrio succinogenes contains molybdenum, iron, and acid-labile sulfide and is also inhibited by cyanide (19). Considerable diversity in electron acceptor specificities exists among FDHs from the anaerobic eubacteria. The highly purified FDHs from C. thermoaceticum (T.

Mechanistic studies have been undertaken on the coenzyme F420 dependent formate dehydrogenase from Methanobacterium formicicum. The enzyme was specific for the si face hydride transfer to C5 of F420 and joins three other F420-recognizing methanogen enzymes in this stereospecificity, consistent perhaps with a common type of binding site for this 8-hydroxy-5-deazariboflavin. While catalysis probably occurs by hydride transfer from formate to the enzyme to generate an EH2 species and then by hydride transfer back out to F420, the formate-derived hydrogen exchanged with solvent protons before transfer back out to F420. The kinetics of hydride transfer from formate revealed that this step is not rate determining, which suggests that the rate-determining step is an internal electron transfer. The deflavo formate dehydrogenase was amenable to reconstitution with flavin analogues. The enzyme was sensitive to alterations in FAD structure in the 6-, 7-, and 8-loci of the benzenoid moiety in the isoalloxazine ring.

No abstract