Purification and characterization of an azoreductase from Escherichia coli CD-2 possessing quinone reductase activity

An oxygen-insensitive intracellular enzyme that is responsible for the decolorization of azo dyes was purified from Escherichia coli CD-2. The molecular weight of the purified enzyme was estimated as 27,000 ± 500 Da. Protein identification indicated that the enzyme had high sequence homology with E. coli K12 quinone reductase, and the enzyme was proved to have both azoreductase and quinone reductase activity. With methyl red as substrate, the optimal pH value and temperature were 6.5 and 37 °C, respectively. The enzyme was stable under different physiochemical conditions. The azoreductase activity was restrained by SDS and was almost completely inhibited by Co²⁺ and Hg²⁺. K_m and V_max values were 0.18 mM and 8.12 U mg^?1 of protein for NADH and 0.05 mM and 6.46 U mg^?1 of protein for methyl red, respectively. The purified enzyme could efficiently decolorize methyl red with both NADH and NADPH as electron donors.     

Assembly of Escherichia coli fumarate reductase holoenzyme

The production and assembly of the four fumarate reductase polypeptides into holoenzyme was studied in vivo in a T7-promoter-conditional expression system. No posttranslational modification of any of the subunits was detected, although the ratio of polypeptides produced varied with the temperature at which expression occurred. FrdC and FrdD, the membrane anchor polypeptides, assembled rapidly into the membrane and then were capped with FrdA and FrdB in separate events. Truncation of the C-terminal domain of FrdD by insertion of transposon Tn5 into the frdD cistron interfered with membrane insertion of the anchor polypeptides and assembly of the holoenzyme. Proteolytic degradation of truncated FrdD was implicated in the production of a soluble FrdABC trimer.     

Succinate dehydrogenase and fumarate reductase from Escherichia coli

Succinate-ubiquinone oxidoreductase (SQR) as part of the trichloroacetic acid cycle and menaquinol-fumarate oxidoreductase (QFR) used for anaerobic respiration by Escherichia coli are structurally and functionally related membrane-bound enzyme complexes. Each enzyme complex is composed of four distinct subunits. The recent solution of the X-ray structure of QFR has provided new insights into the function of these enzymes. Both enzyme complexes contain a catalytic domain composed of a subunit with a covalently bound flavin cofactor, the dicarboxylate binding site, and an iron–sulfur subunit which contains three distinct iron–sulfur clusters. The catalytic domain is bound to the cytoplasmic membrane by two hydrophobic membrane anchor subunits that also form the site(s) for interaction with quinones. The membrane domain of E. coli SQR is also the site where the heme b₅₅₆ is located. The structure and function of SQR and QFR are briefly summarized in this communication and the similarities and differences in the membrane domain of the two enzymes are discussed.     

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.     

Interactions of oxaloacetate with Escherichia coli fumarate reductase

Fumarate reductase of Escherichia coli is converted to a deactivated state when tightly bound by oxaloacetate (OAA). Incubation of the inhibited enzyme with anions or reduction of the enzyme by substrate restores both the activity of the enzyme and its sensitivity to thiol reagents. In these respects the enzyme behaves like cardiac succinate dehydrogenase. Close to an order of magnitude difference was found to exist between the affinities of OAA for the oxidized (KD approximately 0.12 microM) and reduced (KD approximately 0.9 microM) forms of fumarate reductase. Redox titrations of deactivated fumarate reductase preparations have confirmed that reductive activation, as in cardiac succinate dehydrogenase (B. A. C. Ackrell, E. B. Kearney, and D. Edmondson (1975) J. Biol. Chem. 250, 7114-7119), is the result of reduction of the covalently bound FAD moiety and not the non-heme iron clusters of the enzyme. However, the processes differed for the two enzymes; activation of fumarate reductase involved 2e- and 1H+, consistent with reduction of the flavin to the anionic hydroquinone form, whereas the process requires 2e- and 2H+ in cardiac succinate dehydrogenase. The reason for the difference is not known. The redox potential of the FAD/FADH2 couple in FRD (Em approximately -55 mV) was also slightly more positive than that in cardiac succinate dehydrogenase (-90 mV).     

Succinate dehydrogenase and fumarate reductase from Escherichia coli.

Succinate-ubiquinone oxidoreductase (SQR) as part of the trichloroacetic acid cycle and menaquinol-fumarate oxidoreductase (QFR) used for anaerobic respiration by Escherichia coli are structurally and functionally related membrane-bound enzyme complexes. Each enzyme complex is composed of four distinct subunits. The recent solution of the X-ray structure of QFR has provided new insights into the function of these enzymes. Both enzyme complexes contain a catalytic domain composed of a subunit with a covalently bound flavin cofactor, the dicarboxylate binding site, and an iron-sulfur subunit which contains three distinct iron-sulfur clusters. The catalytic domain is bound to the cytoplasmic membrane by two hydrophobic membrane anchor subunits that also form the site(s) for interaction with quinones. The membrane domain of E. coli SQR is also the site where the heme b556 is located. The structure and function of SQR and QFR are briefly summarized in this communication and the similarities and differences in the membrane domain of the two enzymes are discussed.     

Conformational properties of membrane-bound fumarate reductase of Escherichia coli

Anaerobically grown cells of Escherichia coli harboring the plasmid pFRD63 over-produce fumarate reductase, a membrane-bound complex localized in the inner membrane of the cell, where this enzyme represents at least 90% of the total membrane proteins (B. D. Lemire, J. J. Robinson, and J. H. Weiner (1982) J. Bacteriol. 152, 1126-1131). Preparations of inner membrane fractions suspended in 40% sucrose are optically clear, allowing optical spectroscopic measurements. Circular dichroism spectra showed that between pH 6 and 11 the secondary structure of the enzyme is at least 55% in alpha helix and that above pH 11 the structure abruptly changes to a beta-like conformation. The same phenomenon is observed in samples solubilized in the nonionic detergent C12E9. Absorption spectra of the enzyme either membrane bound or solubilized in detergents or exposed to alkaline pH showed that the accessibility of the active site to solvent components is modulated by the interaction of the protein with the membrane. Solubilization of the membrane-bound enzyme with 1% Triton X-100 or C12E9 produced a decrease in ellipticity and in enzymatic activity.     

Electron spin-echo spectroscopic studies of Escherichia coli fumarate reductase

Electron spin-echo envelope modulation (ESEEM) spectroscopy was applied to the study of reduced Centre 1 of Escherichia coli fumarate reductase (succinate:(acceptor) oxidoreductase, EC 1.3.99.1). The ESEEM spectrum derived from stimulated (3-pulse) echo envelopes obtained at 8.8 GHz contained lines at 0.9, 2.1, 3.0 and 4.2 MHz in the g = 1.94 region. When studied at 11.4 GHz, these low-frequency components scale with magnetic field in a manner indicating interaction between the unpaired electron spin of the Fe-S cluster and a weakly coupled 14N nucleus. Spectral simulations of these ESEEM data yield nuclear quadrupole interaction parameters indicative of peptide nitrogen. For oxidized protein, the magnetic-field dependence of the linear electric-field effect (LEFE) for Centre 3 was measured, and the results confirm the presence of a [3Fe-4S] cluster in the protein.     

Isolation and characterization of the tubular organelles induced by fumarate reductase overproduction in Escherichia coli

Strains of Escherichia coli amplifying the intrinsic membrane enzyme fumarate reductase accommodate the overproduced enzyme by increasing the amount of membrane material, in the form of intracellular tubular structures. These tubules have been observed in strains harbouring multicopy frd plasmids and in ampicillin hyper-resistant strains. A procedure has been developed for isolation of tubules nearly free of cytoplasmic membrane. Using protein A-gold labelling and optical diffraction of electron micrographs, a model for tubule structure is proposed. The tubules have a lower lipid/protein ratio than the cytoplasmic membrane, with the enzyme accounting for greater than 90% of the protein in the tubules. Both cytoplasmic membranes and tubules from amplified strains are enriched in cardiolipin and have a more fluid fatty acid composition than wild-type strains. Mutants defective in cardiolipin synthesis produce tubules in response to excess fumarate reductase, but these tubules have an altered appearance, indicating that lipid-protein interactions may be important for tubule assembly.     

Enhancing survival of Escherichia coli by expression of azoreductase AZR possessing quinone reductase activity

Quinone reductase activity of azoreductase AZR from Rhodobacter sphaeroides was reported. High homologies were found in the cofactor/substrate-binding regions of quinone reductases from different domains. 3D structure comparison revealed that AZR shared a common overall topology with mammal NAD(P)H/quinone oxidoreductase NQO1. With menadione as substrate, the optimal pH value and temperature were pH 8-9 and 50 degrees C, respectively. Following the ping-pong kinetics, AZR transferred two electrons from NADPH to quinone substrate. It could reduce naphthoquinones and anthraquinones, such as menadione, lawsone, anthraquinone-2-sulfonate, and anthraquinone-2,6-disulfonate. However, no activity was detected with 1,4-benzoquinone. Dicoumarol competitively inhibited AZR's quinone reductase activity with respect to NADPH, with an obtained K (i) value of 87.6 muM. Significantly higher survival rates were obtained in Escherichia coli YB overexpressing AZR than in the control strain when treated by heat shock and oxidative stressors such as H(2)O(2) and menadione.     

Identification of membrane anchor polypeptides of Escherichia coli fumarate reductase

Fumarate reductase of Escherichia coli has been shown to be a membrane-bound enzyme composed of a 69,000-dalton catalytic-flavin-containing subunit and a 27,000-dalton nonheme-iron-containing subunit. Using gene cloning and amplification techniques, we have observed two additional polypeptides encoded by the frd operon, with apparent molecular weights of 15,000 and 14,000, which are expressed when E. coli is grown anaerobically on glycerol plus fumarate. Expression of these two small polypeptides is necessary for the two large subunits to associate with the membrane. The four subunits remain associated in Triton X-100 extracts of the membrane, and a holoenzyme form of fumarate reductase containing one copy of each of the four polypeptides has been isolated. Unlike the well-characterized two-subunit form, the holoenzyme is not dependent on anions for activity and is not labile at alkaline pH. In these respects, it more closely resembles the membrane-bound activity.     

Wolinella succinogenes quinol:fumarate reductase and its comparison to E. coli succinate:quinone reductase

The three-dimensional structure of Wolinella succinogenes quinol:fumarate reductase (QFR), a dihaem-containing member of the superfamily of succinate:quinone oxidoreductases (SQOR), has been determined at 2.2 A resolution by X-ray crystallography [Lancaster et al., Nature 402 (1999) 377-385]. The structure and mechanism of W. succinogenes QFR and their relevance to the SQOR superfamily have recently been reviewed [Lancaster, Adv. Protein Chem. 63 (2003) 131-149]. Here, a comparison is presented of W. succinogenes QFR to the recently determined structure of the mono-haem containing succinate:quinone reductase from Escherichia coli [Yankovskaya et al., Science 299 (2003) 700-704]. In spite of differences in polypeptide and haem composition, the overall topology of the membrane anchors and their relative orientation to the conserved hydrophilic subunits is strikingly similar. A major difference is the lack of any evidence for a 'proximal' quinone site, close to the hydrophilic subunits, in W. succinogenes QFR.     

Improved conversion of fumarate to succinate by Escherichia coli strains amplified for fumarate reductase

Two recombinant plasmid Escherichia coli strains containing amplified fumarate reductase activity converted fumarate to succinate at significantly higher rates and yields than a wild-type E. coli strain. Glucose was required for the conversion of fumarate to succinate, and in the absence of glucose or in cultures with a low cell density, malate accumulated. Two-dimensional gel electrophoretic analysis of proteins from the recombinant DNA and wild-type strains showed that increased quantities of both large and small fumarate reductase subunits were expressed in the recombinant DNA strains.     

Molybdenum effector of fumarate reductase repression and nitrate reductase induction in Escherichia coli.

In Escherichia coli the presence of nitrate prevents the utilization of fumarate as an anaerobic electron acceptor. The induction of the narC operon encoding the nitrate reductase is coupled to the repression of the frd operon encoding the fumarate reductase. This coupling is mediated by nitrate as an effector and the narL product as the regulatory protein (S. Iuchi and E. C. C. Lin, Proc. Natl. Acad. Sci. USA 84:3901-3905, 1987). The protein-ligand complex appears to control narC positively but frd negatively. In the present study we found that a molybdenum coeffector acted synergistically with nitrate in the regulation of frd and narC. In chlD mutants believed to be impaired in molybdate transport (or processing), full repression of phi(frd-lac) and full induction of phi(narC-lac) by nitrate did not occur unless the growth medium was directly supplemented with molybdate (1 microM). This requirement was not clearly manifested in wild-type cells, apparently because it was met by the trace quantities of molybdate present as a contaminant in the mineral medium. In chlB mutants, which are known to accumulate the Mo cofactor because of its failure to be inserted as a prosthetic group into proteins such as nitrate reductase, nitrate repression of frd and induction of narC were also intensified by molybdate supplementation. In this case a deficiency of the molybdenum coeffector might have resulted from enhanced feedback inhibition of molybdate transport (or processing) by the elevated level of the unutilized Mo cofactor. In addition, mutations in chlE, which are known to block the synthesis of the organic moiety of the Mo cofactor, lowered the threshold concentration of nitrate (< 1 micromole) necessary for frd repression and narC induction. These changes could be explained simply by the higher intracellular nitrate attainable in cells lacking the ability to destroy the effector.     

Improved conversion of fumarate to succinate by Escherichia coli strains amplified for fumarate reductase.

Two recombinant plasmid Escherichia coli strains containing amplified fumarate reductase activity converted fumarate to succinate at significantly higher rates and yields than a wild-type E. coli strain. Glucose was required for the conversion of fumarate to succinate, and in the absence of glucose or in cultures with a low cell density, malate accumulated. Two-dimensional gel electrophoretic analysis of proteins from the recombinant DNA and wild-type strains showed that increased quantities of both large and small fumarate reductase subunits were expressed in the recombinant DNA strains.     

Comparison of catalytic activity and inhibitors of quinone reactions of succinate dehydrogenase (Succinate-ubiquinone oxidoreductase) and fumarate reductase (Menaquinol-fumarate oxidoreductase) from Escherichia coli.

Escherichia coli succinate-ubiquinone oxidoreductase (SQR) and menaquinol-fumarate reductase (QFR) are excellent model systems to understand the function of eukaryotic Complex II. They have structural and catalytic properties similar to their eukaryotic counterpart. An exception is that potent inhibitors of mammalian Complex II, such as thenoyltrifluoroacetone and carboxanilides, only weakly inhibit their bacterial counterparts. This lack of good inhibitors of quinone reactions and the higher level of side reactions in the prokaryotic enzymes has hampered the elucidation of the mechanism of quinone oxidation/reduction in E. coli Complex II. In this communication DT-diaphorase and an appropriate quinone are used to measure quinol-fumarate reductase activity and E. coli bo-oxidase and quinones are used to determine succinate-quinone reductase activity. Simple Michaelis kinetics are observed for both enzymes with ubiquinones and menaquinones in the succinate oxidase (forward) and fumarate reductase (reverse) reactions. The comparison of E. coli SQR and QFR demonstrates that 2-n-heptyl 4-hydroxyquinoline-N-oxide (HQNO) is a potent inhibitor of QFR in both assays; however, SQR is not sensitive to HQNO. A series of 2-alkyl-4,6-dinitrophenols and pentachlorophenol were found to be potent competitive inhibitors of both SQR and QFR. In addition, the isolated E. coli SQR complex demonstrates a mixed-type inhibition with carboxanilides, whereas the QFR complex is resistant to this inhibitor. The kinetic properties of SQR and QFR suggest that either ubiquinone or menaquinone operates at a single exchangeable site working in forward or reverse reactions. The pH activity profiles for E. coli QFR and SQR are similar showing maximal activity between pH 7.4 and 7.8, suggesting the importance of similar catalytic groups in quinol deprotonation and oxidation.     

Structure of fumarate reductase on the cytoplasmic membrane of Escherichia coli.

The terminal electron transfer enzyme fumarate reductase has been shown to be composed of a membrane-extrinsic catalytic dimer of 69- and 27-kilodalton (kd) subunits and a membrane-intrinsic anchor portion of 15- and 13-kd subunits. We prepared inverted membrane vesicles from a strain carrying the frd operon on a multicopy plasmid. When grown anaerobically on fumarate-containing medium, the membranes of this strain are highly enriched in fumarate reductase. When negatively stained preparations of these vesicles were examined with an electron microscope, they appeared to be covered with knob-like structures about 4 nm in diameter attached to the membrane by short stalks. Treatment of the membranes with chymotrypsin destroyed the 69-kd subunit, leaving the 27-, 15-, and 13-kd subunits bound to the membrane; these membranes appeared to retain remnants of the structure. Treatment of the membranes with 6 M urea removed the 69- and 27-kd subunits, leaving the anchor polypeptides intact. These vesicles appeared smooth and structureless. A functional four-subunit enzyme and the knob-like structure could be reconstituted by the addition of soluble catalytic subunits to the urea-stripped membranes. In addition to the vesicular structures, we observed unusual tubular structures which were covered with a helical array of fumarate reductase knobs.     

Investigation of Escherichia coli fumarate reductase subunit function using transposon Tn5

Seventy two Tn5 transposon insertions were isolated in the frd operon carried on the multicopy plasmid pFRD79. The polar nature of these mutations permitted examination of the expression and localization of the frd polypeptides in novel subunit combinations. The minimal catalytic unit is the FRDA plus B dimer. A transposon within frdB (frdB::Tn5) produces inactive, soluble FRDA polypeptide which has covalently attached 8 alpha(N3-histidyl)flavin adenine dinucleotide cofactor. A transposon mutation within frdC (frdC::Tn5) produces soluble, catalytically active dimer. An insertion in frdD (frdD::Tn5) produces both a soluble trimer composed of FRDABC, and a tetramer of FRDABC and truncated FRDD bound to the inner membrane. Eighty percent of the activity is in the soluble form. Using this mutant, the requirement for FRDD both for optimal activity of the catalytic domain and for proper anchorage in the cytoplasmic membrane was demonstrated.     

Differences in protonation of ubiquinone and menaquinone in fumarate reductase from Escherichia coli

Escherichia coli quinol-fumarate reductase operates with both natural quinones, ubiquinone (UQ) and menaquinone (MQ), at a single quinone binding site. We have utilized a combination of mutagenesis, kinetic, EPR, and Fourier transform infrared methods to study the role of two residues, Lys-B228 and Glu-C29, at the quinol-fumarate reductase quinone binding site in reactions with MQ and UQ. The data demonstrate that Lys-B228 provides a strong hydrogen bond to MQ and is essential for reactions with both quinone types. Substitution of Glu-C29 with Leu and Phe caused a dramatic decrease in enzymatic reactions with MQ in agreement with previous studies, however, the succinate-UQ reductase reaction remains unaffected. Elimination of a negative charge in Glu-C29 mutant enzymes resulted in significantly increased stabilization of both UQ-* and MQ-* semiquinones. The data presented here suggest similar hydrogen bonding of the C1 carbonyl of both MQ and UQ, whereas there is different hydrogen bonding for their C4 carbonyls. The differences are shown by a single point mutation of Glu-C29, which transforms the enzyme from one that is predominantly a menaquinol-fumarate reductase to one that is essentially only functional as a succinate-ubiquinone reductase. These findings represent an example of how enzymes that are designed to accommodate either UQ or MQ at a single Q binding site may nevertheless develop sufficient plasticity at the binding pocket to react differently with MQ and UQ.