A periplasmic location for methanol dehydrogenase from Paracoccus denitrificans: implications for proton pumping by cytochrome aa3

Evidence is presented that methanol dehydrogenase from $\text{Paracoccus}\text{denitrificans}$ has a periplasmic location. The implications for the mechanism of proton translocation during electron flow from methanol to oxygen via cytochromes c and aa₃, or to nitrite via cytochrome c and nitrite reductase, are discussed.     

Synthesis and lysis of formate by immobilized cells of Escherichia coli

Formate hydrogenlyase (FHL) activity was induced in a strain of Escherichia coli S13 during anaerobic growth in yeast extract-tryptone medium containing 100 mM formate. The cells obtained at the optimum growth phase were immobilized in 2.5% (w/v) agar gel when 50-60% of the whole cell FHL activity was retained. The immobilized FHL system had good storage stability and recycling efficiency. In the lysis of formate, an increase of formate concentration to 1.18M increased QH(2) (initial) value of the immobilized cell, and subsequently cells, hydrogen evolution, in general, ceased after 6 to 8 of incubation, resulting in incomplete lysis of formate. Presence of small amount of glucose (28 mM) was more or less quantitatively lysed with concomitant disappearence of glucose from the medium. Synthesis of formate from hydrogen and bicarbonate solution by the immobilized cells was also characterized. Presence of glucose (10 mM) in 50 mM bicarbonate solution stimulated formate synthesis by immobilized cells. The pH optimum range, K(m), and specific activity of the immobilized cells for the lysis of formate were 6.8-7.2 0.4M, and 66 mL/g cell-h, respectively. The cells could fix hydrogen to the extent of 24.4% (w/w) of its own wet cell mass in a 72-h reaction cycle. Potentiality of the immobilized FHL system for biotechnological exploitation was discussed.     

Molecular cloning of the fdhF gene of Escherichia coli K-12

Abstract The fdhF gene of Escherichia coli , coding for at least one component of benzyl viologen-linked formate dehydrogenase (FDH-BV) activity, was isolated on a ColE1- fdhF hybrid plasmid from the Clarke and Carbon colony bank.
Endonuclease restriction maps of this plasmid and its pBR322-subcloned derivative, pLW06, were constructed. Various hybrid plasmids were further obtained by deletion of endonuclease-cleaved fragments from pLW06 DNA. Their complementation pattern was analyzed after introduction into different fdhF mutant strains. The fdhF gene was shown to be located on a 5.5 kb Bam HI- Pvu II-DNA fragment, which restored FDH-BV activity to the wild-type level.     

Kinetic and thermodynamic properties of the folding and assembly of formate dehydrogenase

The folding mechanism and stability of dimeric formate dehydrogenase from Candida methylica was analysed by exposure to denaturing agents and to heat. Equilibrium denaturation data yielded a dissociation constant of about 10⁻¹³ M for assembly of the protein from unfolded chains and the kinetics of refolding and unfolding revealed that the overall process comprises two steps. In the first step a marginally stable folded monomeric state is formed at a rate (k₁) of about 2 × 10⁻³ s⁻¹ (by deduction k₋₁ is about10⁻⁴ s⁻¹) and assembles into the active dimeric state with a bimolecular rate constant (k₂) of about 2 × 10⁴ M⁻¹ s⁻¹. The rate of dissociation of the dimeric state in physiological conditions is extremely slow (k₋₂ ∼ 3 × 10⁻⁷ s⁻¹).     

The organization of formate dehydrogenase in the cytoplasmic membrane of Escherichia coli.

The arrangement of the proton-translocating formate dehydrogenase of the anaerobic respiratory chain of Escherichia coli within the cytoplasmic membrane was examined by direct covalent modification with non-membrane-permeant reagents. Three methods were employed, lactoperoxidase-catalysed radioiodination, labelling with diazotized [125I] di-iodosulphanilic acid and labelling with diazobenzene [35S] sulphonate. All three procedures yield consistent with the view that the two larger subunits of the enzyme, Mr 110000 and 32000, both occupy transmembranous locations within the membrane. In each case the modification of the Ca2+ or Mg2+-activated F1-ATPase was monitored, and all reagents employed correctly located this enzyme at the cytoplasmic face of the membrane. A procedure involving agglutination with specific antibodies is described which appears to fractionate membrane vesicles of mixed orientation into two populations, one with the same membrane orientation as that of spheroplasts and the other opposite orientation.     

Escherichia coli formate-hydrogen lyase. Purification and properties of the selenium-dependent formate dehydrogenase component

The formate-hydrogen lyase complex of Escherichia coli decomposes formic acid to hydrogen and carbon dioxide under anaerobic conditions in the absence of exogenous electron acceptors. The complex consists of two separable enzymatic activities: a formate dehydrogenase and a hydrogenase. The formate dehydrogenase component (FDHH) of the formate-hydrogen lyase complex was purified to near homogeneity in two column chromatographic steps. The purified enzyme was composed of a single polypeptide of molecular weight 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Metal analysis showed each mole of enzyme contained 3.3 g atoms of iron. Denaturation of FDHH released a compound which, when oxidized, displayed a fluorescence spectrum similar to that of the molybdopterin cofactor found in certain other enzymes. The enzyme contained selenium in the form of selenocysteine as determined by radioactive labeling of the enzyme with 75Se and amino acid analysis. FDHH activity was maximal between pH 7.5 and 8.5; however, the enzyme was maximally stable at pH 5.3-6.4 and highly unstable above pH 7.5. Nitrate and nitrite salts caused a drastic reduction in activity. Although azide inhibited FDHH activity, it also protected the enzyme from inactivation by oxygen.     

Estimating the energetic contribution of hydrogen bonding to the stability of Candida methylica formate dehydrogenase by using double mutant cycle

An homology model of Candida methylica formate dehydrogenase (cm FDH) was constructed based on the Pseudomonas sp. 101 formate dehydrogenase (ps FDH) structure. In wild type cm FDH, Thr169 and Thr226 can form hydrogen bonds with each other. We measured the interaction energy between the two threonines independent of other interactions in the proteins by using a so-called double mutant cycle and assessing the protein stability from the concentration of guanidine hydrochloride needed to denature 50% of the molecules. We conclude that the hydrogen bonds stabilize the wild type protein by -4 kcal mol(-1).     

Inactive and temperature-sensitive folding mutants generated by tryptophan substitutions in the membrane-bound d-lactate dehydrogenase of Escherichia coli

A combination of site-specific mutagenesis and 19F nuclear magnetic resonance has been used to investigate the structural properties of D-lactate dehydrogenase, a membrane-associated enzyme of Escherichia coli. The protein (65,000 Da) has been labeled with 5-fluorotryptophan for 19F nuclear magnetic resonance studies. Tryptophan has been substituted for individual phenylalanine, tyrosine, isoleucine, and leucine residues at various positions throughout the enzyme molecule, and the fluorinated native and substituted tryptophan residues have been used as probes of the local environment. All 24 mutants thus generated are expressed in E. coli. Ten are fully active and purfiable following the usual procedure, while 14 either are inactive or produce low levels of activity. The amount of active enzyme produced from the low-yield mutants is dependent on the temperature at which synthesis is carried out, with more active enzyme produced at 18 degrees C than at 27, 35, or 42 degrees C. Cells grown at 27 degrees C and then incubated at 42 degrees C retain 90-100% of their activity. All of the expressed protein from the inactive mutants is Triton-insoluble, aggregated, and not readily purfiable; the inactive mutant protein appears to be improperly folded. Most of the expressed D-lactate dehydrogenase from the partially active mutants is also Triton-insoluble; a small fraction, however, is soluble in Triton and can be purified to yield active enzyme. All the purified enzymes from these low-yield mutants of D-lactate dehydrogenase have essentially normal VmaxS, and all but two have normal KmS. Once purified, the low-yield mutant enzymes are stable at 42 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the fdhF gene encoding the selenopolypeptide for benzyl viologen-linked formate dehydrogenase in Escherichia coli

Summary In Podospora anserina a chromosome walk near the mating type locus was made possible through isolation of genomic sequences linked to a plasmid integrated in this part of the genome. Genetic analysis of 86 transformants obtained from the 5 first cosmids of this walk was performed. These data and those reported elsewhere for cosmids resulting from another chromosome walk allow us to draw two clear-cut rules for transformation with cosmids. First, the large majority of transformants arise from integration at the resident locus, contrasting with the heterologous process which predominates for plasmids. Second, all homologous integrations are highly unstable while all non-homologous integrations are stable. Analysis of the timing of the instability reveals that loss of the selective marker is probably limited to the fruiting body.     

Nuclear magnetic resonance and molecular genetic studies of the membrane-bound D-lactate dehydrogenase of Escherichia coli

In this study we demonstrate the potential of combining fluorine-19 nuclear magnetic resonance (NMR) spectroscopy with molecular genetics. We are using the membrane-bound enzyme D-lactate dehydrogenase of Escherichia coli as a model system to characterize interactions between proteins and lipids. We have labeled D-lactate dehydrogenase with 4-, 5-, and 6-fluorotryptophans and obtained high-resolution fluorine-19 NMR spectra showing five resonances, in agreement with the five tryptophan residues expected from the DNA sequence. The five 19F resonances in the spectra have been assigned to the specific tryptophan residues in the primary sequence of D-lactate dehydrogenase by site-directed oligonucleotide mutagenesis of the cloned gene. We observe large differences in the relative fluorine-19 chemical shifts of each tryptophan residue when labeled by different isomers of fluorotryptophan. We have determined by NMR methods that two tryptophans are exposed to the solvent and that none of the tryptophan residues are within 10 A of the lipid phase. On the basis of 19F NMR spectroscopy of the labeled tryptophan residues, the conformation of D-lactate dehydrogenase is similar in aqueous solution and in the presence of a variety of lipids and detergents. This result indicates that the presence of lipids or detergents is not required to maintain the tertiary structure of this membrane-bound enzyme. In contrast, Triton X-100 induces a change to an abnormal conformation of the enzyme as judged from both NMR spectroscopy and the effect of temperature on the maximal velocity of the enzyme in the presence of this detergent.     

Proton translocation by the F1F0ATPase of Escherichia coli. Mutagenic analysis of the a subunit

Cassette site-directed mutagenesis was employed to generate mutations in the a subunit (uncB (a) gene) of F1F0ATP synthase. Using sequence homology with similar subunits of other F1F0ATP synthases as a guide, 20 mutations were targeted to a region of the a subunit thought to constitute part of the proton translocation mechanism. ATP-driven proton pumping activity is lost with the substitution of lys, ile, val, or glu for arginine 210. Substitution of val, leu, gln, or glu for asparagine 214 does not completely block proton conduction, however, replacement of asparagine 214 with histidine does reduce enzyme activity below that necessary for significant function. Two or three mutations were constructed in each of four nonpolar amino acids, leucine 207, leucine 211, alanine 217, and glycine 218. Certain specific mutations in these positions result in partial loss of F1F0ATP synthase activity, but only the substitution of arginine for alanine 217 reduces ATP-driven proton pumping activity to undetectable levels. It is concluded that of the six amino acids studied, only arginine 210 is an essential component of the proton translocation mechanism. Fractionation of cell-free extracts of a subunit mutation strains generally reveals normal amounts of F1 specifically bound to the particulate fraction. One possible exception is the arginine 210 to isoleucine mutation which results in somewhat elevated levels of free F1 detectable in the soluble fraction. For nearly all a subunit mutations, F1F0-mediated ATP hydrolysis activity remains sensitive to inhibition by dicyclohexylcarbodiimide in spite of the fact that the mutations block proton translocation.     

Residues in the Polar Loop of Subunit c in Escherichia coli ATP Synthase Function in Gating Proton Transport to the Cytoplasm

Rotary catalysis in F₁F₀ ATP synthase is powered by proton translocation through the membrane-embedded F₀ sector. Proton binding and release occur in the middle of the membrane at Asp-61 on the second transmembrane helix (TMH) of subunit c, which folds in a hairpin-like structure with two TMHs. Previously, the aqueous accessibility of Cys substitutions in the transmembrane regions of subunit c was probed by testing the inhibitory effects of Ag⁺ or Cd²⁺ on function, which revealed extensive aqueous access in the region around Asp-61 and on the half of TMH2 extending to the cytoplasm. In the current study, we surveyed the Ag⁺ and Cd²⁺ sensitivity of Cys substitutions in the loop of the helical hairpin and used a variety of assays to categorize the mechanisms by which Ag⁺ or Cd²⁺ chelation with the Cys thiolates caused inhibition. We identified two distinct metal-sensitive regions in the cytoplasmic loop where function was inhibited by different mechanisms. Metal binding to Cys substitutions in the N-terminal half of the loop resulted in an uncoupling of F₁ from F₀ with release of F₁ from the membrane. In contrast, substitutions in the C-terminal half of the loop retained membrane-bound F₁ after metal treatment. In several of these cases, inhibition was shown to be due to blockage of passive H⁺ translocation through F₀ as assayed with F₀ reconstituted into liposomes. The results suggest that the C-terminal domain of the cytoplasmic loop may function in gating H⁺ translocation to the cytoplasm.     

Effects of solubilisation on some properties of the membrane-bound respiratory enzyme D-amino acid dehydrogenase of Escherichia coli

Solubilisation, delipidation and partial purification of the membrane-bound enzyme D-amino acid dehydrogenase of Escherichia coli K12 produced significant changes in several of its properties. Solubilised enzyme showed a broader substrate specificity, increased affinity for at least three substrates, and a lower pH optimum with D-alanine as substrate. Solubilised enzyme was more heat-labile than native enzyme, particularly at 37 degrees C, and re-binding to envelope preparations restored protection against heat denaturation. Activity of delipidated enzyme could be increased by addition of pure phospholipids. Native enzyme showed biphasic Arrhenius kinetics associated with phase changes of membrane lipids.     

Proton motive force-dependent and -independent protein translocation revealed by an efficient in vitro assay system of Escherichia coli

Inverted membrane vesicles prepared from Escherichia coli spheroplasts were fractionated by means of sucrose gradient centrifugation, and a vesicle preparation exhibiting efficient and quantitative translocation of secretory proteins was obtained. The translocation of OmpA and an uncleavable model protein, uncleavable OmpF-Lpp, took place almost completely in 2-3 min, whereas that of OmpF-Lpp, a chimeric secretory protein, required 20 min for completion. The requirement of the proton motive force (delta muH+) for in vitro translocation was then examined with these three proteins. The translocation of all these proteins was significantly inhibited by the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or when stripped membrane vesicles lacking F1-ATPase were used, suggesting that delta muH+ generally participates in the translocation reaction. The inhibition was complete with OmpF-Lpp, whereas significant amounts of uncleavable OmpF-Lpp and OmpA were translocated at a slower rate even with the stripped membrane vesicles in the presence of a high concentration of carbonyl cyanide m-chlorophenylhydrazone. The delta muH+-independent translocation was inhibited by a nonhydrolyzable ATP analogue. These results indicate that although translocation of OmpF-Lpp obligatory requires delta muH+, the latter two proteins can be translocated in not only a delta muH+-dependent manner but also a delta mu H+-independent manner.     

Inhibition of the membrane-bound adenosine triphosphatase of Escherichia coli by dicyclohexylcarbodi-imide.

The inhibition of the membrane-bound adenosine triphosphatase of Escherichia coli by DCCD (dicyclohexylcarbodi-imide) is studied under conditions of varying KCl concentration. An increase in K+ concentration and in other cations causes an increase in the DCCD sensitivity of the enzyme, as well as significant changes in the kinetic parameters.