Identification of molybdopterin guanine dinucleotide in formate dehydrogenase from Methanobacterium formicicum.

The pterin cofactor in formate dehydrogenase isolated from Methanobacterium formicium is identified as molybdopterin guanine dinucleotide. The pterin, stabilized as the alkylated, dicarboxamidomethyl derivative, is shown to have absorption and chromatographic properties identical to those of the previously characterized authentic compound. Treatment with nucleotide pyrophosphatase produced the expected degradation products GMP and carboxyamidomethyl molybdopterin. The molybdopterin guanine dinucleotide released from the enzyme by treatment with 95% dimethyl sulfoxide is shown to be functional in the in vitro reconstitution of the cofactor-deficient nitrate reductase in extracts of the Neurospora crassa nit-1 mutant.

Mechanistic studies of the coenzyme F420 reducing formate dehydrogenase from Methanobacterium formicicum.

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.

Cloning, expression, and nucleotide sequence of the formate dehydrogenase genes from Methanobacterium formicicum.

The genes for the two subunits of the formate dehydrogenase from Methanobacterium formicicum were cloned and their sequences determined. When expressed in Escherichia coli, two proteins were produced which had the appropriate mobility on an SDS gel for the two subunits of formate dehydrogenase and cross-reacted with antibodies raised to purified formate dehydrogenase. The genes for the two formate dehydrogenase subunits overlap by 1 base pair and are preceded by DNA sequences similar to both eubacterial and archaebacterial promoters and ribosome-binding sites. The amino acid sequences deduced from the DNA sequence were analyzed, and the arrangement of putative iron-sulfur centers is discussed.

Molybdopterin cofactor from Methanobacterium formicicum formate dehydrogenase.

The molybdopterin cofactor from the formate dehydrogenase of Methanobacterium formicicum was studied. The cofactor was released by guanidine denaturation of homogeneous enzyme, which also released greater than 80% of the molybdenum present in the enzyme. The anoxically isolated cofactor was nonfluorescent, but after exposure to air it fluoresced with spectra similar to those of described molybdopterin cofactors. Aerobic release from acid-denatured formate dehydrogenase in the presence of I2 and potassium iodide produced a mixture of fluorescent products. Alkaline permanganate oxidation of the mixture yielded pterin-6-carboxylic acid as the only detectable fluorescent product. The results showed that the cofactor from formate dehydrogenase contained a pterin nucleus with a 6-alkyl side chain of unknown structure. Covalently bound phosphate was also present. The isolated cofactor was unable to complement the cofactor-deficient nitrate reductase of the Neurospora crassa nit-1 mutant.

Composition of the coenzyme F420-dependent formate dehydrogenase from Methanobacterium formicicum.

The coenzyme F420-dependent formate dehydrogenase from Methanobacterium formicicum was purified to electrophoretic homogeneity by anoxic procedures which included the addition of azide, flavin adenine dinucleotide (FAD), glycerol, and 2-mercaptoethanol to all buffer solutions to stabilize activity. The enzyme contains, in approximate molar 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 alpha 1 beta 1. The molecular weight of the alpha-subunit is 85,000, and that of the beta-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.

Formate dehydrogenase from Methanobacterium formicicum. Electron paramagnetic resonance spectroscopy of the molybdenum and iron-sulfur centers.

Formate dehydrogenase from Methanobacterium formicicum was examined by electron paramagnetic resonance spectroscopy. Although oxidized enzyme yielded no EPR signals over the temperature range 8-200 K, dithionite reduction resulted in generation of two paramagnetic components. The first, a nearly isotropic signal visible at temperatures below 200 K with g1 = 2.018, g2 = 2.003, and g3 = 1.994, exhibited nuclear hyperfine interaction with two equivalent protons (A1 = 0.45, A2 = 0.6, and A3 = 0.55 milliTeslas). EPR spectra of partially reduced 95Mo-enriched formate dehydrogenase exhibited additional 3-4 milliTeslas splittings, due to spin interaction with the 95Mo nucleus. Thus, this signal is due to a Mo center. This is the first reported example of a Mo center with gav greater than 2.0 in a biological system. The second species, a rhombic signal visible below 40 K with g values of g1 = 2.0465, g2 = 1.9482, and g3 = 1.9111 showed no hyperfine coupling and was assigned to reduced Fe/S. Both paramagnetic species could be detected in samples of M. formicicum whole cells anaerobically reduced with sodium formate. The Mo(V) signal was altered following addition of cyanide (g1 = 1.996, g2 = 1.988, and g3 = 1.980). Growth of bacteria in the presence of 1 mM WO4(2-) resulted in abolition of the Mo(V) EPR signal and formate dehydrogenase activity. Em, 7.7 was -330 mV for Mo(VI)/Mo(V) and -470 mV for Mo(V)/Mo(IV).

FAD requirement for the reduction of coenzyme F420 by formate dehydrogenase from Methanobacterium formicicum.

The partial purification of the formate dehydrogenase from cell-free extracts of Methanobacterium formicicum decreased the rate of coenzyme F420 reduction 175-fold relative to the rate of methyl viologen reduction. FAD, isolated from this organism, reactivated the coenzyme F420-dependent activity of purified formate dehydrogenase and restored the activity ratio (coenzyme F420/methyl viologen) to near that in cell-free extracts. Neither flavin mononucleotide nor FADH2 replaced FAD. The reduced form of FAD inhibited the reactivation of coenzyme F420-dependent formate dehydrogenase activity by the oxidized form. The results suggest that native formate dehydrogenase from Methanobacterium formicicum contains noncovalently bound FAD that is required for coenzyme F420-dependent activity.

Kinetics of Formate Metabolism in Methanobacterium formicicum and Methanospirillum hungatei.

The kinetics of formate metabolism in Methanobacterium formicicum and Methanospirillum hungatei were studied with log-phase formate-grown cultures. The progress of formate degradation was followed by the formyltetrahydrofolate synthetase assay for formate and fitted to the integrated form of the Michaelis-Menten equation. The K(m) and V(max) values for Methanobacterium formicicum were 0.58 mM formate and 0.037 mol of formate h g (dry weight), respectively. The lowest concentration of formate metabolized by Methanobacterium formicicum was 26 muM. The K(m) and V(max) values for Methanospirillum hungatei were 0.22 mM and 0.044 mol of formate h g (dry weight), respectively. The lowest concentration of formate metabolized by Methanospirillum hungatei was 15 muM. The apparent K(m) for formate by formate dehydrogenase in cell-free extracts of Methanospirillum hungatei was 0.11 mM. The K(m) for H(2) uptake by cultures of Methanobacterium formicicum was 6 muM dissolved H(2). Formate and H(2) were equivalent electron donors for methanogenesis when both substrates were above saturation; however, H(2) uptake was severely depressed when formate was above saturation and the dissolved H(2) was below 6 muM. Formate-grown cultures of Methanobacterium formicicum that were substrate limited for 57 h showed an immediate increase in growth and methanogenesis when formate was added to above saturation.

Properties of formate dehydrogenase in Methanobacterium formicicum.

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 mumol of methyl viologen per min per mg of protein and 8.2 mumol 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 microM) than for coenzyme F420 (6 microM). The purified enzyme also reduced flavin mononucleotide (Km = 13 microM) and flavin adenine dinucleotide (Km = 25 microM) 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 degrees C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 microM), azide (Ki = 39 microM), alpha,alpha-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.

Metabolism of formate in Methanobacterium formicicum.

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 muM. 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 degrees 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 degrees 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 degrees 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.

A modified blow-gun syringe for remote injection of captive wildlife.

A modified syringe capable of automatic injection and suitable for use with a blow-gun is described. The syringe has been used successfully with white-tailed deer (Odocoileus virginianus) under confined conditions. Desirable characteristics for blow-gun syringes are discussed.

Heavy metal levels in feathers of wild turkeys from Virginia.

Levels of lead, cadmium, nickel, and zinc were determined in feathers of 175 wild turkeys (Meleagris gallopava) shot by hunters in 19 Virginia counties in 2 physiographic regions. Lead and nickel levels did not vary by county, region, sex, or age. Zinc and cadmium levels were significantly (P less than 0.01) higher in adult turkeys.