Genetic analysis of mutants of Salmonella typhimurium deficient in formate dehydrogenase activity

Summary A genetical study of mutants of Salmonella typhimurium deficient in formate dehydrogenase activity was performed. The affected gene was designated fdh A and mapped at 116 min, the order of genes in that region being xyl-fdh A-mtl-cys E.Abbreviations FHL formate hydrogenylase - FDH (PMS) formate dehydrogenase (phenazine methosulfate) - FDH (BV) formate dehydrogenase (benzyl viologen) - HYD hydrogenase - NR nitrate-reductase - TTR tetrathionate-reductase     

Nitrate reductase complex of Escherichia coli K-12: participation of specific formate dehydrogenase and cytochrome b1 components in nitrate reduction

The participation of distinct formate dehydrogenases and cytochrome components in nitrate reduction by Escherichia coli was studied. The formate dehydrogenase activity present in extracts prepared from nitrate-induced cells of strain HfrH was active with various electron acceptors, including methylene blue, phenazine methosulfate, and benzyl viologen. Certain mutants which are unable to reduce nitrate had low or undetectable levels of formate dehydrogenase activity assayed with methylene blue or phenazine methosulfate as electron acceptor. Of nine such mutants, five produced gas when grown anaerobically without nitrate and possessed a benzyl viologen-linked formate dehydrogenase activity, suggesting that distinct formate dehydrogenases participate in the nitrate reductase and formic hydrogenlyase systems. The other four mutants formed little gas when grown anaerobically in the absence of nitrate and lacked the benzyl viologen-linked formate dehydrogenase as well as the methylene blue or phenazine methosulfate-linked activity. The cytochrome b(1) present in nitrate-induced cells was distinguished by its spectral properties and its genetic control from the major cytochrome b(1) components of aerobic cells and of cells grown anaerobically in the absence of nitrate. The nitrate-specific cytochrome b(1) was completely and rapidly reduced by 1 mm formate but was not reduced by 1 mm reduced nicotinamide adenine dinucleotide; ascorbate reduced only part of the cytochrome b(1) which was reduced by formate. When nitrate was added, the formate-reduced cytochrome b(1) was oxidized with biphasic kinetics, but the ascorbate-reduced cytochrome b(1) was oxidized with monophasic kinetics. The inhibitory effects of n-heptyl hydroxyquinoline-N-oxide on the oxidation of cytochrome b(1) by nitrate provided evidence that the nitrate-specific cytochrome is composed of two components which have different redox potentials but identical spectral properties. We conclude from these studies that nitrate reduction in E. coli is mediated by the sequential operation of a specific formate dehydrogenase, two specific cytochrome b(1) components, and nitrate reductase.     

Escherichia coli formate dehydrogenase mutants with altered selenopolymer profiles

Four classes of Escherichia coli mutants deficient in either or both of their anaerobic selenium-containing formate dehydrogenases (FDH) were isolated. A class I mutant devoid of FDH_H activity specifically linked to benzyl viologen (BV) produced a small amount of the FDH_H 80,000 dalton selenopeptide. Three class II mutants were deficient in FDH_N activity specifically linked to phenazine methosulfate (PMS) and exhibited a selenopeptide doublet rather than the FDH_N 110,000 dalton selenosubunit. Three class III mutants were selenium incorporation deficient and did not exhibit either FDH activity or ⁷⁵Selabeled selenopolymers. A class IV mutant was devoid of PMS-linked FDH_N activity; neither its FDH_N 110,000 dalton selenosubunit nor its BV-linked FDH_H activity was fully regulated by nitrate.Abbreviations FDH formate dehydrogenase - BV benzyl viologen - MV methyl viologen - PMS phenazine methosulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

A physiological study of formate dehydrogenase,formate oxidase and hydrogenlyase fromEscherichia coli K-12

Escherichia coli was grown under various culture conditions. Variations in the levels of formate dehydrogenase which reacts with methylene blue (MB) or phenazine methosulfate (PMS) (N enzyme), formate dehydrogenase which reacts with benzyl viologen (BV) (H enzyme), formate oxidase and hydrogenlyase were analyzed. It was observed that formate dehydrogenase N and formate oxidase were induced by nitrate and repressed by oxygen. Synthesis of formate dehydrogenase H and hydrogenlyase was induced by formate and repressed by nitrate and oxygen. Selenite was required for the biosynthesis of formate dehydrogenase H and hydrogenlyase. Activity of both formate oxidase and hydrogenlyase was inhibited by azide and KCN but not by N-heptyl hydroxyquinoline-N-oxide (HOQNO); on the other hand, formate oxidase was extremely sensitive to HOQNO. Data were obtained which suggest that cytochromes are not involved in hydrogen formation from formate. Part of this work was carried out when the senior author was visiting Research Biologist in the Laboratory of Dr. J. A. de Mosss at the University of California, San Diego. Thanks are given to Dr. De Moss for his hospitality and advise and to Dr. Warren Butler of the University of California, San Diego for making available his spectrophotometer to carry out cytochrome analyses. Most of this work was sustained by a grant from the Research Corporation, Brown Hazen Fund and the financial help of the C.O.F.A.A. from the Instituto Politécnico Nacional.     

Some properties of formate dehydrogenase,accumulation and incorporation of 185W-tungsten into proteins of Clostridium formicoaceticum

Formate dehydrogenase of Clostridium formicoaceticum used only methyl and benzyl viologen, but not NAD as electron acceptor. The S_0.5 values were 0.9×10^-4 M for formate and 5.8×10^-3 M for methyl viologen. Using potassium phosphate buffer a pH-optimum of 7.9 was observed. The initial velocity of the formate dehydrogenase activity reached a maximum at 70°C, whereas the activity was stable only up to 50°C. The level of formate dehydrogenase in C. formicoaceticum was increased to its maximum when 10^-6 M selenite and 10^-7 M tungstate were added to a synthetic medium. Addition of molybdate instead of tungstate did not increase the level of formate dehydrogenase. ¹⁸⁵W-tungsten was concentrated about 100-fold by C. formicoaceticum; molybdate had no major effect on the uptake of tungsten. ¹⁸⁵W-tungsten was found almost exclusively in the soluble fluid and was predominantly recovered after chromatography in a protein of about 88000 molecular weight. Occasionally a labelled protein of low molecular weight was observed. Again molybdate added even in high molar excess did not influence the labelling pattern. No radioactivity peak could be obtained at the elution peak of formate dehydrogenase activity. The extreme instability of formate dehydrogenase prevented further purification.Abbreviations FDH formate dehydrogenase - DTE dithioerythritol - HEPES hydroxyethylpiperazine N-2-ethane sulconic acid - TEA triethylamine - DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulfate - TTC triphenyltetrazolium     

Studies on nitrate reductase from Cyanidium caldarium

Summary A nitrate reductase from the thermophilic acidophilic alga, Cyanidium caldarium, was studied. The enzyme utilises the reduced forms of benzyl viologen and flavins as well as both NADPH₂ and NADH₂ as electron donors to reduce nitrate.Heat treatment has an activating effect on the benzyl viologen (FMNH₂, FADH₂) nitrate reductase. At 50°C the activation of the enzyme is complete in about 20 min of exposure, whereas at higher temperatures (until 75°C) it is virtually an instantaneous phenomenon. The observed increase in activity is very low in extracts from potassium nitrate grown cells, whereas it is 5 or more fold in extracts from ammonium sulphate supplied cells. The benzyl viologen nitrate reductase is stable at 60°C and is destroyed at 75°C after 3 min; the NADPH₂ nitrate reductase is destroyed at 60°C. The pH optimum for both activities was found in the range 7.8–8.2.Ammonium nitrate grown cells possess a very low level of nitrate reductase: when they are transferred to a nitrate medium a rapid synthesis of enzyme occurs. By contrast, when cells with fully induced activity are supplied with ammonia, a rapid loss of NADPH₂ and benzyl viologen nitrate reductase occurs; however, activity measured with heated extracts shows that the true level of benzyl viologen nitrate reductase is as high as before ammonium addition. It is suggested that the presence of ammonia causes a rapid inactivation but no degradation of the enzyme.Cycloheximide inhibits the formation of the enzyme; the drug is without effect on the loss of nitrate reductase activity induced by ammonium. The nitrate reductase is reactivated in vivo by the removal of the ammonium, in the absence as well as in the presence of cycloheximide.     

The thermodynamic properties of some commonly used oxidation-reduction mediators,inhibitors and dyes,as determined by polarography

The oxidation-reduction midpoint potentials (E_m) of the following compounds have been measured in the range of pH from 3 to 12 by polarography: methyl viologen; benzyl viologen; 2-hydroxy-1,4-naphthoquinone; 2-hydroxy-1,4-anthraquinone; N,N,N′,N′,-tetramethyl-p-phenylenediamine;2,3,5,6-tetramethyl-p-phenylenediamine; phenazine; N-methylphenazonium methosulfate; N-methylphenazonium sulfonate methosulfate; N-ethylphenazonium ethosulfate; pyocyanine; neutral red; safranin; phenol red; chlorophenol red; cresol red; bromocresol purple; 2,5-dibromo-3-methyl-6-isopropylbenzoquinone and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole. Many of these previously assumed to have a simple behavior in this range have proven to be rather more complicated, and several anomalous observations have been reconciled.     

Resolution of distinct selenium-containing formate dehydrogenases from Escherichia coli.

Formate dehydrogenase, a component activity of two alternative electron transport pathways in anaerobic Escherichia coli, has been resolved as two distinguishable enzymes. One, which was induced with nitrate reductase as a component of the formate-nitrate reductase pathway, utilized phenazine methosulfate (PMS) in preference to benzyl viologen (BV) as an artificial electron acceptor and appeared to be exclusively membrane-bound. A second formate dehydrogenase, which was induced as a component of the formate hydrogenlyase pathway, appeared to exist both as a membrane-bound form and as a cytoplasmic enzyme; the cytoplasmic activity was resolved completely from the PMS-linked activity on a sucrose gradient. When E. coli was grown in the presence of 75Se-selenite, a 110,000-dalton selenopeptide, previously shown to be a component of the PMS-linked enzyme, was induced and repressed with this activity. In contrast, an 80,000-dalton selenopeptide was induced and repressed with the BV-linked activity and exhibited a distribution similar to the BV-linked formate dehydrogenase in cell fractions and in sucrose gradients. The results indicate that the two formate dehydrogenases are distinguishable on the basis of their artificial electron acceptor specificity, their cellular localization, and the size of their respective selenoprotein components.     

Isolation and characterization of mutant strains of Escherichia coli altered in H2 metabolism

A positive selection procedure is described for the isolation of hydrogenase-defective mutant strains of Escherichia coli. Mutant strains isolated by this procedure can be divided into two major classes. Class I mutants produced hydrogenase activity (determined by using a tritium-exchange assay) and formate hydrogenlyase activity but lacked the ability to reduce benzyl viologen or fumarate with H2 as the electron donor. Class II mutants failed to produce active hydrogenase and hydrogenase-dependent activities. All the mutant strains produced detectable levels of formate dehydrogenase-1 and -2 and fumarate reductase. The mutation in class I mutants mapped near 65 min of the E. coli chromosome, whereas the mutation in class II mutants mapped between srl and cys operons (58 and 59 min, respectively) in the genome. The class II Hyd mutants can be further subdivided into two groups (hydA and hydB) based on the cotransduction characteristics with cys and srl. These results indicate that there are two hyd operons and one hup operon in the E. coli chromosome. The two hyd operons are needed for the production of active hydrogenase, and all three are essential for hydrogen-dependent growth of the cell.     

[1-14C] Acetate assimilation by obligate methylotrophs,Pseudomonas methanica and Methylosinus trichosporium

The oxidation of one carbon compounds (methane, methanol, formaldehyde, formate) and primary alcohols (ethanol, propanol, butanol) supported the assimilation of [1-¹⁴C]acetate by cell suspensions of type I obligate methylotroph; Pseudomonas methanica, Texas strain, and type II obligate methylotroph, Methylosinus trichosporium, strain PG. The amount of oxygen consumed and substrate oxidized correlated with the amount of [1-¹⁴C]acetate assimilated during oxidation of C-1 compounds and primary alcohols.Oxidation of methanol, formaldehyde, and primary alcohols in extracts of Pseudomonas methanica, Texas strain, and Methylosinus trichosporium, strain PG, was catalyzed by a phenazine methosulfate linked, ammonium ion dependent methanol dehydrogenase. The oxidation of aldehydes was catalyzed by a phenazine methosulfate linked, ammonium ion independent aldehyde dehydrogenase. Formate was oxidized by a NAD⁺ linked formate dehydrogenase.Deceased.This work was supported by Grant GB 8173 from the National Science Foundation and by a grant from the Robert A. Welch Foundation.     

Localization and characterization of cytochromes from membrane vesicles of Escherichia coli K-12 grown in anaerobiosis with nitrate

Cytochromes b of anaerobically nitrate-grown Escherichia coli cells are analysed. Ascorbate phenazine methosulfate distinguishes low and high potential cytochromes b. Reduction kinetics performed at 559 nm presents a very complex pattern which can be analysed assuming that at least four b-type cytochromes are present. The electron transport chain from formate to oxygen would contain a low potential cytochrome b-556, a cytochrome b-558 associated to the oxidase, and a cytochrome d as the principal oxidase. Cytochrome o is also present, but seems to be functional only at low oxygen concentrations. A cytochrome b-556 associated to nitrate reductase is shown to belong to a branch of the formate-oxidase chain.2-N-Heptyl-4-hydroxyquinoline-N-oxide affects the reduction kinetics in a very complex way. One inhibition site is in evidence between cytochrome b-558 and cytochrome d; another between the cytochrome associated to nitrate reductase and the nitrate reductase. A third inhibition site is located in the common part of the formate-nitrate and the formate-oxidase systems.Ascorbate phenazine methosulfate is shown to donate electrons near cytochrome b-558.     

Localization and characterization of cytochromes from membrane vesicles of Escherichia coli K-12 grown in anaerobiosis with nitrate.

Cytochromes b of anaerobically nitrate-grown Escherichia coli cells are analysed. Ascorbate phenazine methosulfate distinguishes low and high potential cytochromes b. Reduction kinetics performed at 559 nm presents a very complex pattern which can be analysed assuming that at least four b-type cytochromes are present. The electron transport chain from formate to oxygen would contain a low potential cytochrome b-556, a cytochrome b-558 associated to the oxidase, and a cytochrome d as the principle oxidase. Cytochrome o is also present, but seems to be functional only at low oxygen concentrations. A cytochrome b-556 associated to nitrate reductase is shown to belong to a branch of the formate-oxidase chain. 2-N-Heptyl-4-hydroxyquinoline-N-oxide affects the reduction kinetics in a very complex way. One inhibition site is in evidence between cytochrome b-558 and cytochrome d; another between the cytochrome associated to nitrate reductase and the nitrate reductase. A third inhibition site is located in the common part of the formate-nitrate and the formate-oxidase systems. Ascorbate phenazine methosulfate is shown to donate electrons near cytochrome b-558.     

Genetic and physiological characterization of new Escherichia coli mutants impaired in hydrogenase activity

The Mu dl (ApR lac) bacteriophage was used to generate mutants of Escherichia coli which were defective in formate hydrogenlyase. Three mutants were chosen for further analysis: they lacked hydrogenase (hydrogen: benzyl viologen oxidoreductase) activity, but produced normal levels of fumarate reductase activity and two- to three-fold reduced levels of benzyl viologen (BV)-dependent formate dehydrogenase activity. Two of them (hydC) were shown to contain about 4-fold reduced amounts of formate hydrogenlyase and fumarate-dependent H2 uptake activities. The third one (hydD) was totally devoid of both activities. Their insertion sites were located at 77 min on the E. coli map. Subdivision of these mutants into two classes was subsequently based on the restoration capacity of hydrogenase activity with high concentration of nickel in the growth media. Addition of 500 microM NiCl2 led to a complete recovery of hydrogenase activity, and to the concomitant restoration of normal BV-linked formate dehydrogenase, formate hydrogenlyase and fumarate-dependent H2 uptake activities in the hydC mutants. The hydD mutant was insensitive to the effect of nickel. Expression of the lac operon in hydC and hydD mutants was induced by anaerobiosis. It was not increased by the addition of formate under anaerobic conditions. The presence of nitrate resulted in slightly reduced beta-galactosidase activities in the hydC mutants, whereas those found in the hydD mutant reached only one third of the level obtained in its absence. Fumarate had no effect on both classes. Moreover, in contrast to the hydD locus, the hydC::Mu dl fusions were found to be dependent upon the positive control exerted by the nirR gene product and were totally repressed by an excess of nickel. In addition, the low levels of overall hydrogenase-dependent activities found in a nirR strain were also relieved by the presence of nickel. Our results strongly suggest that the pleiotropic regulatory gene nirR is essential for the expression of a gene (hydC) involved in either transport or processing of nickel in the cell, whose alteration leads to a loss of hydrogenase activity.     

Electron donors and the quinone involved in dimethyl sulfoxide reduction inEscherichia coli

Escherichia coli can use dimethyl sulfoxide (DMSO) as an electron acceptor during anaerobic growth on the oxidizable substrate, glycerol. During growth, the DMSO is reduced to dimethyl sulfide (DMS). For the reduction of DMSO, NADH, formate, lactate, reduced benzyl viologen, reduced methyl viologen, and dithionite can serve as electron donors. The terminal reductase and the dehydrogenases linking the various electron donors to the electron transport chain were found to be membrane bound. Chlorate-resistant mutants (chl) were unable to grow and reduce DMSO. However, in the case of thechlD mutant, growth and DMSO reduction can be restored by growth in the presence of high concentrations of molybdate. Mutants ofE. coli blocked in menaquinone (vitamin K₂) biosynthesis—menB, menC, andmenD—were unable to grow with DMSO as an electron acceptor, even though the terminal reductase is present in these mutants. Both growth and DMSO reduction could be restored in these mutants by growth in the presence of the menaquinone intermediates,o-succinylbenzoate and 1,4-dihydroxy-2-naphthoate, depending on the metabolic block of the mutant. Thus menaquinone is involved in electron transport during DMSO reduction.     

Soluble Dissimilatory Nitrate Reductase Containing Cytochrome c from a Photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

Dissimilatory nitrate reductase [nitrite: (acceptor) oxidoreductase.EC 1.7.99.4 [EC] ] from a denitrifying photosynthetic bacterium, Rhodopseudomonassphaeroides forma sp. denitrificans proved to be a soluble enzymethat could be purified 47-fold. It was labile, and containedcytochrome c, based on the results of specific staining forheme on polyacrylamide gel electrophoresis and on its absorptionspectrum. Its physiological molecular weight was determinedto be 112k, although heterogeneous molecular weights of 112k,100k, 73k and 60k were found for different preparations. Theoptimum for enzyme activity was about pH 6, and the K_m for thenitrate was 1.6 mM. As an electron donor, benzyl viologen wasvery good; but NADH, NADPH, FAD, FMN, cytochromes b₂ and c₂,dichlorophenolindophenol and phenazine methosulfate were noteffective. Bathophenanthroline and thiocyanate inhibited enzymaticactivity. The addition of 1 mM tungstate to the growing culturein place of molybdate decreased the nitrate reductase in thecells, but a further addition of 1 mM molybdate stopped it.This nitrate reductase is believed to be a molybdo-iron proteinsimilar to the enzymes from other bacteria with a nitrate respiratingability. (Received February 29, 1980; Accepted January 29, 1981)     

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (carboxydebacteria) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H₂-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H₂ with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD⁺, FMN⁺, and NAD(P)⁺ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H₂OCO₂+2H⁺+2e^- as previously shown for Pseudomonas carboxydovorans.Dedicated to Otto Kandler remembering almost three decades of enjoyable cooperation     

The purification and properties of a cd-cytochrome nitrite reductase from Paracoccus halodenitrificans

Paracoccus halodenitrificans, grown anaerobically in the presence of nitrite, contained membrane and cytoplasmic nitrite reductases. When assayed in the presence of phenazine methosulfate and ascorbate, the membranebound enzyme produced nitrous oxide whereas the cytoplasmic enzyme produced nitric oxide. When both enzymes were assayed in the presence of methyl viologen and dithionite, the cytoplasmic enzyme produced ammonia. Following solubilization, the membrane-bound enzyme behaved like the cytoplasmic enzyme, producing nitric oxide in the presence of phenazine methosulfate and ascorbate, and ammonia when assayed in the presence of methyl viologen and dithionite. The cytoplasmic and membranebound enzymes were purified to essentially the same specific activity. Only a single nitrite-reductase activity was detected on electrophoretic gels and the electrophoretic behavior of both enzymes suggested they were identical. The spectral properties of both enzymes suggested they were cd-type cytochromes. These data suggest that the products of nitrite reduction by the cd-cytochrome nitrite reductase are determined by the location of the enzyme and the redox potential of the electron donor.Abbreviations PMS phenazine methosulfate - MV methyl viologen - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid - CHAPSO [3-(3-cholamidopropyldimethylammonia)-1-(2-hydroxy-1-propanesulfonate)] National Research Council Research Fellow     

Anaerobiosis induces expression of ant, a new Escherichia coli locus with a role in anaerobic electron transport

Escherichia coli has a formate hydrogenlyase system which allows it to maintain an electron balance during anaerobic growth by passing electrons from formate to H+ ions, thus generating H2. The Mu d1(Ap lac) bacteriophage was used to generate mutants that were defective in passing electrons from formate to benzyl viologen, an artificial electron acceptor. A subset of these mutants was studied in which beta-galactosidase was expressed at much higher levels under anaerobic conditions than under aerobic conditions. If nitrate was present during anaerobic growth, the same levels of beta-galactosidase were seen in these fusion strains as were seen under aerobic conditions. The Mu d1(Ap lac) insertions in these mutants were genetically mapped between mutS and srl and thus define a new locus we have termed ant (anaerobic electron transport). Recombinant lambda derivatives were isolated which complemented the deficiency of the ant mutants in anaerobic electron transport and also carried a trans-acting region of DNA which reduced expression of the ant-lac fusions under anaerobic conditions; a probe to the ant region was generated from one of these recombinant lambda derivatives. Southern hybridization analysis revealed that the four independent ant::Mu d1(Ap lac) fusions we isolated spanned an approximately 5-kilobase region and that all were transcribed in the same direction, counterclockwise on the E. coli genetic map.     

Kinetics for formate dehydrogenase of Escherichia coli formate-hydrogenlyase

Kinetic parameters of the selenium-containing, formate dehydrogenase component of the Escherichia coli formate-hydrogenlyase complex have been determined with purified enzyme. A ping-pong Bi Bi kinetic mechanism was observed. The Km for formate is 26 mM, and the Km for the electron-accepting dye, benzyl viologen, is in the range 1-5 mM. The maximal turnover rate for the formate-dependent catalysis of benzyl viologen reduction was calculated to be 1.7 x 10(5) min-1. Isotope exchange analysis showed that the enzyme catalyzes carbon exchange between carbon dioxide and formate in the absence of other electron acceptors, confirming the ping-pong reaction mechanism. Dissociation constants for formate (12.2 mM) and CO2 (8.3 mM) were derived from analysis of the isotope exchange data. The enzyme catalyzes oxidation of the alternative substrate deuterioformate with little change in the Vmax, but the Km for deuterioformate is approximately three times that of protioformate. This implies formate oxidation is not rate-limiting in the overall coupled reaction of formate oxidation and benzyl viologen reduction. The deuterium isotope effect on Vmax/Km was observed to be approximately 4.2-4.5. Sodium nitrate was found to inhibit enzyme activity in a competitive manner with respect to formate, with a Ki of 7.1 mM. Sodium azide is a noncompetitive inhibitor with a Ki of about 80 microM.     

Formate hydrogenlyase system in Salmonella typhimurium LT2.

Isolation from Salmonella typhimurium of mutants unable to reduce benzyl viologen under anaerobic conditions has allowed the study of the factors involved in the multienzymic formate hydrogenylase system. 1. Depending on the affected activities, different classes of mutants were found: FHL-A mutants have lost formate dehydrogenase 1 and formate dehydrogenase 2 activities; mutations in fdhA (117 min) or fdhB (33 min) lead to such a phenotype. FHL-B and FHL-C mutants have lost formate dehydrogenase 2 activity and part or all of hydrogenase activity, respectively; both types correspond to mutations in the hyd gene (approximately 90 min). FHL-D mutants have lost only formate dehydrogenase 2 activity; fhlD gene maps at 120 min. 2. In some cases, mixtures of extracts from two mutants display formate dehydrogenase 2 and formate hydrogenylase activities. Restoration studies suggest the existence of one factor sensitive to growth conditions and inactivated by oxygen or heating. This factor which is present and active in FHL-C mutants, is probably the one missing in FHL-D mutants. 3. A new scheme for the formate hydrogenylase system is proposed, in which hydrogenase transfers electrons directly to benzyl viologen.