Localization of dehydrogenases, reductases, and electron transfer components in the sulfate-reducing bacterium Desulfovibrio gigas.

Various dehydrogenases, reductases, and electron transfer proteins involved in respiratory sulfate reduction by Desulfovibrio gigas have been localized with respect to the periplasmic space, membrane, and cytoplasm. This species was grown on a lactate-sulfate medium, and the distribution of enzyme activities and concentrations of electron transfer components were determined in intact cells, cell fractions prepared with a French press, and lysozyme spheroplasts. A significant fraction of formate dehydrogenase was demonstrated to be localized in the periplasmic space in addition to hydrogenase and some c-type cytochrome. Cytochrome b, menaquinone, fumarate reductase, and nitrite reductase were largely localized on the cytoplasmic membrane. Fumarate reductase was situated on the inner aspect on the membrane, and the nitrite reductase appeared to be transmembraneous. Adenylylsulfate reductase, bisulfite reductase (desulfoviridin), pyruvate dehydrogenase, and succinate dehydrogenase activities were localized in the cytoplasm. Significant amounts of hydrogenase and c-type cytochromes were also detected in the cytoplasm. Growth of D. gigas on a formate-sulfate medium containing acetate resulted in a 10-fold increase in membrane-bound formate dehydrogenase and a doubling of c-type cytochromes. Growth on fumarate with formate resulted in an additional increase in b-type cytochrome compared with lactate-sulfate-grown cells.     

Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli.

Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic-lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM. Two peptides with molecular weights of 70 000 and 24 000, predent in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Stephacryl S-200 gel filtration in the presence of sodium cholate. This would indicate that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.     

Regulation of hydrogenase activity in enterobacteria.

Proteus vulgaris, Escherichia coli, and Citrobacter freundii cells were devoid of hydrogenase activity when grown on complex medium or minimal medium plus glucose in the presence of saturating levels of dissolved oxygen. Anaerobically grown cells had appreciable hydrogenase activity. Cells grown anaerobically in the presence of CO (an inhibitor of hydrogenase) or nitrate (an electron acceptor) lacked hydrogenase activity. To make hydrogenase essential for anaerobic growth, cells were grown on fumarate, a nonfermentable carbon source. P. vulgaris and C. freundii evolved H2 gas under these conditions, and the hydrogenase-specific activity was 8 to 10 times greater than that in cells grown on glucose. Cell growth was inhibited by CO, and the cells grew but lacked hydrogenase activity when grown in the presence of nitrate. E. coli grew on fumarate plus H2, and the specific activity was five times greater than that in cells grown on glucose. Thus, hydrogenase activity is inducible and is expressed maximally when the enzyme is essential for cellular growth. Under conditions of growth where the enzyme would not be catalytically active, cells contain little active hydrogenase. Under anaerobic conditions where the enzyme is not essential for growth, the level of hydrogenase activity is intermediate.     

Effects of Molybdate and Selenite on Formate and Nitrate Metabolism in Escherichia coli

The effects of adding molybdate and selenite to a glucose-minimal salts medium on the formation of enzymes involved in the anaerobic metabolism of formate and nitrate in Escherichia coli have been studied. When cells were grown anaerobically in the presence of nitrate, molybdate stimulated the formation of nitrate reductase and a b-type cytochrome, resulting in cells that had the capacity for active nitrate reduction in the absence of formate dehydrogenase. Under the same conditions, selenite in addition to molybdate was required for forming the enzyme system which permits formate to serve as an effective electron donor for nitrate reduction. When cells were grown anaerobically on a glucose-minimal salts medium without nitrate, active hydrogen production from formate as well as formate dehydrogenase activity depended on the presence of both selenite and molybdate. The effects of these metals on the formation of formate dehydrogenase was blocked by chloramphenicol, suggesting that protein synthesis is required for the increases observed. It is proposed that the same formate dehydrogenase is involved in nitrate reduction, hydrogen production, and in aerobic formate oxidation.     

Mutants of Escherichia coli with altered hydrogenase activity

Mutant strains of Escherichia coli which expressed different levels of hydrogenase activity when grown anaerobically under a variety of conditions were obtained by mutagenesis and selective growth and screening procedures. Four classes of mutants were isolated, ranging from those devoid of enzyme activity to those expressing maximal activity under all growth conditions. One class of mutants (A) could not grow on fumarate plus H2 in the presence of active fumarate reductase. Since hydrogenase is essential for growth under these conditions some of these strains may be hydrogenase-negative. Three other classes of mutants were isolated which were all hydrogenase-positive and fully expressed this activity when grown on fumarate plus H2. They differed in the level of expression of hydrogenase activity when grown anaerobically on glucose, conditions which do not require hydrogenase for growth. Class B mutants expressed less activity, while class C mutants expressed more activity than the parental strain. Class D mutants fully expressed hydrogenase activity and were dependent on the enzyme for growth. The different strains were also assayed for reduction of dyes by hydrogen and for evolution of hydrogen from reduced methyl viologen. Some of the hydrogenase-positive strains showed altered activities in these assays suggesting that mutations may have occurred either in enzymes or proteins required for reaction with dyes or in the hydrogenase enzyme itself.     

Proton translocation associated with anaerobic transhydrogenation from glycerol 3-phosphate to fumarate in Escherichia coli.

An $\text{Escherichia}\text{coli}$ strain, that lacks aerobic glycerol 3-phosphate (G3P) dehydrogenase and succinate dehydrogenase, was grown anaerobically on glycerol and fumarate. The addition of fumarate to such cells resulted in the formation of dihydroxyacetone phosphate (DHAP) suggesting that G3P is oxidized to DHAP while fumarate is reduced to succinate. Associated with the transhydrogenation was an extrusion of protons from the cell into the incubation medium. The stoichiometry of protons extruded to DHAP formed was near 2. Everted membrane vesicles take up protons in the presence of added G3P and fumarate.     

Arabinan degrading enzymes from Aspergillus nidulans: Induction and purification

Abstract The expression and distribution of ferric reductase activity was examined in Shewanella putrefaciens MR-1. Formate-dependent ferric reductase was not detected in aerobically grown cells but was readily detectable in anaerobically grown cells. Ferric reductase activity was found exclusively in the membrane fractions, with 54–56% in the outer membrane. In contrast, the majority of formate dehydrogenase was in the soluble fraction with lesser amounts associated with the various membrane fractions. Outer membrane ferric reductase activity was markedly inhibited by p -chloromercuriphenylsulfonate, 2-heptyl-4-hydroxyquinolone- N -oxide, and antimycin A, but was unaffected by the presence of alternate electron acceptors (nitrate, nitrite, fumarate, and trimethylamine N -oxide). Both formate and NADH served as electron donors for ferric reductase; activity with l -lactate or NADPH was poor. The addition of FMN markedly stimulated formate- and NADH-dependent ferric reductase.     

A second phenazine methosulphate-linked formate dehydrogenase isoenzyme in Escherichia coli.

A biochemical and immunological study has revealed a new formate dehydrogenase isoenzyme in Escherichia coli. The enzyme is an isoenzyme of the respiratory formate dehydrogenase (FDH-N) which forms part of the formate to nitrate respiratory pathway found in the organisms when it is grown anaerobically in the presence of nitrate. The new enzyme, termed FDH-Z, cross reacts with antibodies raised to FDH-N and possesses a similar polypeptide composition to FDH-N. FDH-Z catalyses the phenazine methosulphate-linked formate dehydrogenase activity present in the aerobically-grown bacterium. FDH-Z and FDH-N exhibit distinct regulation. Like formate dehydrogenase N, formate dehydrogenase Z is a membrane-bound molybdoenzyme. With nitrate reductase it can catalyse electron transfer between formate and nitrate. Quinones are required for the physiological electron transfer to nitrate. It seems likely that like FDH-N, FDH-Z functions physiologically as a formate: quinone oxidoreductase.     

Sites and specificity of the reaction of bipyridylium compounds with anaerobic respiratory enzymes of Escherichia coli. Effects of permeability barriers imposed by the cytoplasmic membrane

The ability of the oxidized and singly reduced species of several bipyridylium cations to cross the cytoplasmic membrane of Escherichia coli was studied to locate the sites of reaction of the dyes with anaerobic respiratory enzymes. Benzyl Viologen radical crossed the membrane rapidly, whereas the oxidized species did not. The oxidized or radical species of Methyl Viologen, Morfamquat or Diquat did not rapidly cross the membrane. It was also shown that the dithionite anion does not cross the cytoplasmic membrane of E. coli. Diquat radical donates electrons to the nitrate reductase pathway at the periplasmic aspect of the membrane, whereas Benzyl Viologen radical reacted directly with nitrate reductase itself (EC 1.7.99.4) at the cytoplasmic aspect of the membrane. Thus the pathway of electron transfer in the nitrate reductase pathway is transmembranous. Formate hydrogenlyase (EC 1.2.1.2) and an uncharacterized nitrite reductase activity react with bipyridylium dyes at the periplasmic aspect of the membrane. Fumarate reductase (succinate dehydrogenase; EC 1.3.99.1) reacts with bipyridylium radicals, and formate dehydrogenase (cytochrome) (EC 1.2.2.1) with ferricyanide, at the cytoplasmic aspect of the membrane. The differing charge and membrane permeation of oxidized and radical species of bipyridylium dyes greatly complicate their use as potentiometric mediators in suspensions of cells or membrane vesicles.     

Energy transformation coupled to formate oxidation during anaerobic fermentation

A correlation between the rate of ATP synthesis by F0F1 ATP-synthase and formate oxidation by formate hydrogen lyase (FHL) has been established in inverted membrane vesicles of Escherichia coli JW 136 mutant with double deletions (delta hya/ delta hyb) of hydrogenase 1 and 2 grown anaerobically on glucose in the absence of external electron acceptors (pH 6.5). ATP synthesis was suppressed by H+ -ATPase inhibitors N,N'-dicyclohexylcarbodiimide (DCCD) and sodium azide as well as by the protonophore carbonyl cyanide-m-chlorophenyhydrazone (CCCP). Copper ions inhibited formate-dependent hydrogenase and ATP-synthase activities but did not affect the ATPase activity of vesicles. The maximal rate of ATP synthesis (0.83 microM/min x mg protein) stimulated by K+ ions was determined when sodium formate, ADP and inorganic phosphate were applied simultaneously. The results confirm the assumption about the dual role of hydrogenase 3, formate hydrogen lyase subunit, which is able to couple the reduction of protons to H2 and their translocation through a membrane with chemiosmotic synthesis of ATP.     

Amino acid transport in membrane vesicles of obligately anaerobic Veillonella alcalescens.

Membrane vesicles of Veillonella alcalescens, grown in the presence of L-lactate and KNO-3, actively transport amino acids under anaerobic conditions in the presence of several electron donors and the electron acceptor nitrate. The highest initial rates of uptake are obtained with L-lactate, followed by reduced nicotinamide adenine dinucleotide, glycerol-1-phosphate, formate, and L-malate.. The membrane vesicles contain the dehydrogenases for these electron donors, and these enzymes are coupled with nitrate reductase. In membrane vesicles from cells, grown in the presence of nitrate, the dehydrogenases are not coupled with fumarate reducatase, and anaerobic transport of amino acids does not occur with fumarate as electron acceptor. Under aerobic conditions none of the physiological electron donors can energize transport. However, a high rate of uptake is observed with the electron donor system ascorbate-phenazine metho-sulfate. This electron donor system also effectively energizes transport under anaerobicconditions in the presence of the electron acceptor nitrate.     

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.     

Aerobic inactivation of fumarate reductase from Escherichia coli by mutation of the [3Fe-4S]-quinone binding domain.

Fumarate reductase from Escherichia coli functions both as an anaerobic fumarate reductase and as an aerobic succinate dehydrogenase. A site-directed mutation of E. coli fumarate reductase in which FrdB Pro-159 was replaced with a glutamine or histidine residue was constructed and overexpressed in a strain of E. coli lacking a functional copy of the fumarate reductase or succinate dehydrogenase complex. The consequences of these mutations on bacterial growth, assembly of the enzyme complex, and enzymatic activity were investigated. Both mutations were found to have no effect on anaerobic bacterial growth or on the ability of the enzyme to reduce fumarate compared with the wild-type enzyme. The FrdB Pro-159-to-histidine substitution was normal in its ability to oxidize succinate. In contrast, however, the FrdB Pro-159-to-Gln substitution was found to inhibit aerobic growth of E. coli under conditions requiring a functional succinate dehydrogenase, and furthermore, the aerobic activity of the enzyme was severely inhibited upon incubation in the presence of its substrate, succinate. This inactivation could be prevented by incubating the mutant enzyme complex in an anaerobic environment, separating the catalytic subunits of the fumarate reductase complex from their membrane anchors, or blocking the transfer of electrons from the enzyme to quinones. The results of these studies suggest that the succinate-induced inactivation occurs by the production of hydroxyl radicals generated by a Fenton-type reaction following introduction of this mutation into the [3Fe-4S] binding domain. Additional evidence shows that the substrate-induced inactivation requires quinones, which are the membrane-bound electron acceptors and donors for the succinate dehydrogenase and fumarate reductase activities. These data suggest that the [3Fe-4S] cluster is intimately associated with one of the quinone binding sites found n fumarate reductase and succinate dehydrogenase.     

Variation in proton donor/acceptor pathways in succinate:quinone oxidoreductases

The anaerobically expressed fumarate reductase and aerobically expressed succinate dehydrogenase from Escherichia coli comprise two different classes of succinate:quinone oxidoreductases (SQR), often termed respiratory complex II. The X-ray structures of both membrane-bound complexes have revealed that while the catalytic/soluble domains are structurally similar the quinone binding domains of the enzyme complexes are significantly different. These results suggest that the anaerobic and aerobic forms of complex II have evolved different mechanisms for electron and proton transfer in their respective membrane domains.     

Energy transduction by electron transfer via a pyrrolo-quinoline quinone-dependent glucose dehydrogenase in Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus (var. lwoffi).

The coupling of membrane-bound glucose dehydrogenase (EC 1.1.99.17) to the respiratory chain has been studied in whole cells, cell-free extracts, and membrane vesicles of gram-negative bacteria. Several Escherichia coli strains synthesized glucose dehydrogenase apoenzyme which could be activated by the prosthetic group pyrrolo-quinoline quinone. The synthesis of the glucose dehydrogenase apoenzyme was independent of the presence of glucose in the growth medium. Membrane vesicles of E. coli, grown on glucose or succinate, oxidized glucose to gluconate in the presence of pyrrolo-quinoline quinone. This oxidation led to the generation of a proton motive force which supplied the driving force for uptake of lactose, alanine, and glutamate. Reconstitution of glucose dehydrogenase with limiting amounts of pyrrolo-quinoline quinone allowed manipulation of the rate of electron transfer in membrane vesicles and whole cells. At saturating levels of pyrrolo-quinoline quinone, glucose was the most effective electron donor in E. coli, and glucose oxidation supported secondary transport at even higher rates than oxidation of reduced phenazine methosulfate. Apoenzyme of pyrrolo-quinoline quinone-dependent glucose dehydrogenases with similar properties as the E. coli enzyme were found in Acinetobacter calcoaceticus (var. lwoffi) grown aerobically on acetate and in Pseudomonas aeruginosa grown anaerobically on glucose and nitrate.     

Fumarate reduction in Proteus mirabilis.

1. Proteus mirabilis formed fumarate reductase under anaerobic growth conditions. The formation of this reductase was repressed under conditions of growth during which electron transport to oxygen or to nitrate is possible. In two of three tested chlorate-resistant mutant strains of the wild type, fumarate reductase appeared to be affected. 2. Cytoplasmic membrane suspensions isolated from anaerobically grown P. mirabilis oxidized formate and NADH with oxygen and with fumarate, too. 3. Spectral investigation of the cytoplasmic membrane preparation revealed the presence of (probably at least two types of) cytochrome b, cytochrome a1 and cytochrome d. Cytochrome b was reduced by NADH as well as by formate to approximately 80%. 4. 2-n-Heptyl-4-hydroxyquinilone-N-oxide and antimycin A inhibited oxidation of both formate and NADH by oxygen and fumarate. Both inhibitors increased the level of the formate/oxygen steady state and the formate/fumarate steady state. 5. The site of inhibition of the respiratory activity by both HQNO and antimycin A was located at the oxidation side of cytochrome b. 6. The effect of ultraviolet-irradiation of cytoplasmic membrane suspensions on oxidation/reduction phenomena suggested that the role of menaquinone is more exclusive in the formate/fumarate pathway than in the electron transport route to oxygen. 7. Finally, the conclusion has been drawn that the preferential route for electron transport from formate and from NADH to fumarate (and to oxygen) includes cytochrome b as a directly involved carrier. A hypothetical scheme for the electron transport in anaerobically grown P. mirabilis is presented.     

Proteins of the Inner Membrane of Escherichia coli: Changes in Composition Associated with Anaerobic Growth and Fumarate Reductase Amber Mutation

The inner membrane fractions of Escherichia coli grown anaerobically and aerobically were isolated, and their proteins were compared by electrophoresis in polyacrylamide gels. To maximimize the differences between the preparations, the anaerobic cultures were grown on complex medium with added glucose, but glucose was omitted from the aerobic cultures to prevent catabolite repression. The pattern of bands in the two types of preparation differed considerably, and changes in approximately 20 components were observed. In particular, the band identified as succinate dehydrogenase in aerobic preparations was greatly reduced in anaerobic preparations. Mutants lacking fumarate reductase were isolated, and inner membrane preparations of an frd amber mutant were deficient in a major component of 75,000 daltons and possibly a minor one of 87,500 daltons. The former was also present in greater amounts in anaerobic preparations and could represent a fumarate reductase subunit.     

l-Aspartate fermentation by a free-livingCampylobacter species

In the fermentation ofl-aspartate by a free-livingCampylobacter spec., the products formed were acetate, succinate, carbon dioxide and ammonia. The oxidative part of the fermentation pathway yielded acetate, succinate, carbon dioxide and ammonia, and the reductive part gave rise to the formation of succinate and ammonia. When grown anaerobically with aspartate, cells contained cytochromesb andc as well as menaquinone. Reduced cytochromeb, but not reduced cytochromec could be reoxidized by fumarate. In the presence of nitrate, 90% of the available electrons were transferred to nitrate, which was reduced to nitrite; the remainder was transported via the fumarate reductase system. Cells grown with aspartate and excess of formate converted aspartate quantitatively to succinate.Abbreviation Used TLC thin layer chromatography     

Investigation of the fumarate metabolism of the syntrophic propionate-oxidizing bacterium strain MPOB

The growth of the syntrophic propionate-oxidizing bacterium strain MPOB in pure culture by fumarate disproportionation into carbon dioxide and succinate and by fumarate reduction with propionate, formate or hydrogen as electron donor was studied. The highest growth yield, 12.2 g dry cells/mol fumarate, was observed for growth by fumarate disproportionation. In the presence of hydrogen, formate or propionate, the growth yield was more than twice as low: 4.8, 4.6, and 5.2 g dry cells/mol fumarate, respectively. The location of enzymes that are involved in the electron transport chain during fumarate reduction in strain MPOB was analyzed. Fumarate reductase, succinate dehydrogenase, and ATPase were membrane-bound, while formate dehydrogenase and hydrogenase were loosely attached to the periplasmic side of the membrane. The cells contained cytochrome c, cytochrome b, menaquinone-6 and menaquinone-7 as possible electron carriers. Fumarate reduction with hydrogen in membranes of strain MPOB was inhibited by 2-(heptyl)-4-hydroxyquinoline-N-oxide (HOQNO). This inhibition, together with the activity of fumarate reductase with reduced 2,3-dimethyl-1,4-naphtoquinone (DMNH₂) and the observation that cytochrome b of strain MPOB was oxidized by fumarate, suggested that menequinone and cytochrome b are involved in the electron transport during fumarate reduction in strain MPOB. The growth yields of fumarate reduction with hydrogen or formate as electron donor were similar to the growth yield of Wolinella succinogenes. Therefore, it can be assumed that strain MPOB gains the same amount of ATP from fumarate reduction as W. succinogenes, i.e. 0.7 mol ATP/mol fumarate. This value supports the hypothesis that syntrophic propionate-oxidizing bacteria have to invest two-thirds of an ATP via reversed electron transport in the succinate oxidation step during the oxidation of propionate. The same electron transport chain that is involved in fumarate reduction may operate in the reversed direction to drive the energetically unfavourable oxidation of succinate during syntrophic propionate oxidation since (1) cytochrome b was reduced by succinate and (2) succinate oxidation was similarly inhibited by HOQNO as fumarate reduction. Received: 18 March 1997 / Accepted: 10 November 1997