Quaternary structure and composition of squash NADH:nitrate reductase

NADH:nitrate reductase (EC 1.6.6.1) was isolated from squash cotyledons (Cucurbita maxima L.) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on Bio-Gel A-1.5m. These preparations gave a single protein staining band (Mr = 115,000) on sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is homogeneous. The native Mr of nitrate reductase was found to be 230,000, with a minor form of Mr = 420,000 also occurring. These results indicate that the native nitrate reductase is a homodimer of Mr = 115,000 subunits. Acidic amino acids predominate over basic amino acids, as shown both by the amino acid composition of the enzyme and an isoelectric point for nitrate reductase of 5.7. The homogeneous nitrate reductase had a UV/visible spectrum typical of a b-type cytochrome. The enzyme was found to contain one each of flavin (as FAD), heme iron, molybdenum, and Mo-pterin/Mr = 115,000 subunit. A model is proposed for squash nitrate reductase in which two Mr = 115,000 subunits are joined to made the native enzyme. Each subunit contains 1 eq of FAD, cytochrome b, and molybdenum/Mo-pterin.     

Further characterization of trimethylamine N-oxide reductase from Escherichia coli, a molybdoprotein

Escherichia coli trimethylamine N-oxide (TMAO) reductase I, the major enzyme among inducible TMAO reductases, was purified to homogeneity by an improved method including heat treatment, ammonium sulfate precipitation, and chromatographies on Bio-Gel A-1.5m, DEAE-cellulose, and Reactive blue-agarose. The molecular weight was estimated by gel filtration to be approximately 200,000. A single subunit peptide with a molecular weight of 95,000 was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme contained 1.96 atoms of molybdenum, 0.96 atoms of iron, 1.52 atoms of zinc, and less than 0.4 atoms of acid-labile sulfur per molecular weight of 200,000. The absorption spectrum of the enzyme showed a peak at 278 nm and a shoulder at 288 nm, but no characteristic absorption was found from 350 to 700 nm. A fluorescent derivative of molybdenum cofactor was found when the enzyme was boiled with iodine in acidic solution; its fluorescence spectra were almost the same as those of the form A derivative of molybdopterin found in sulfite oxidase. The molybdenum cofactor released from heated TMAO reductase I reconstituted nitrate reductase in the extracts of Neurospora crassa mutant strain nit-1 lacking molybdenum cofactor. Thus, TMAO reductase I contains molybdopterin, which is a common constituent of some molybdenum-containing enzymes. Some kinetic properties were also determined.     

Escherichia coli nitrate reductase subunit A: its role as the catalytic site and evidence for its modification.

Subunits A and B were isolated from purified nitrate reductase by preparative electrophoresis in low levels of sodium dodecyl sulfate. Nonheme iron and low levels of molybdenum were associated with isolated subunit A but not with isolated subunit B. After dialysis against a source of molybdenum cofactor, subunit A regained tightly bound molybdenum and concomitantly regained enzyme activity and reactivity with anti-nitrate reductase antiserum. Subunit B neither bound cofactor nor regained activity or reactivity with antiserum. These data indicate that subunit A contains the active site of the enzyme. Subunit A was also found to be modified posttranslationally in a similar fashion as is subunit B. This was determined by comparison of partial proteolytic digests and amino acid analyses of A subunits from precursor and membrane-bound forms of nitrate reductase.     

Purification and properties of Escherichia coli dimethyl sulfoxide reductase, an iron-sulfur molybdoenzyme with broad substrate specificity.

Dimethyl sulfoxide reductase, a terminal electron transfer enzyme, was purified from anaerobically grown Escherichia coli harboring a plasmid which codes for dimethyl sulfoxide reductase. The enzyme was purified to greater than 90% homogeneity from cell envelopes by a three-step purification procedure involving extraction with the detergent Triton X-100, chromatofocusing, and DEAE ion-exchange chromatography. The purified enzyme was composed of three subunits with molecular weights of 82,600, 23,600, and 22,700 as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight was determined by gel electrophoresis to be 155,000. The purified enzyme contained 7.5 atoms of iron and 0.34 atom of molybdenum per mol of enzyme. The presence of molybdopterin cofactor in dimethyl sulfoxide reductase was identified by reconstitution of cofactor-deficient NADPH nitrate reductase activity from Neurospora crassa nit-I mutant and by UV absorption and fluorescence emission spectra. The enzyme displayed a very broad substrate specificity, reducing various N-oxide and sulfoxide compounds as well as chlorate and hydroxylamine.     

Purification and properties of succinate-ubiquinone oxidoreductase complex from Paracoccus denitrificans

Highly active succinate-ubiquinone reductase has been purified from cytoplasmic membranes of aerobically grown Paracoccus denitrificans. The purified enzyme has a specific activity of 100 units per mg protein, and a turnover number of 305 s-1. Succinate-ubiquinone reductase activity of the purified enzyme is inhibited by 3'-methylcarboxin and thenoyltrifluoroacetone. Four subunits, with apparent molecular masses of 64.9, 28.9, 13.4 and 12.5 kDa, were observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme contains 5.62 nmol covalently bound flavin and 3.79 nmol cytochrome b per mg protein. The 64.9 kDa subunit was shown to be a flavoprotein by its fluorescence. Polyclonal antibodies raised against this protein cross-reacted with the flavoprotein subunit of bovine heart mitochondrial succinate-ubiquinone reductase. The 28.9 kDa subunit is likely analogous to the bovine heart iron protein, and the cytochrome b heme is probably associated with one or both of the low-molecular-weight polypeptides. The cytochrome b is not reducible with succinate but is reoxidized with fumarate after prereduction with dithionite. Iron-sulfur clusters S-1 and S-3 of the Paracoccus oxidoreductase exhibit EPR spectra very similar to their mitochondrial counterparts. Paracoccus succinate-ubiquinone reductase complex is thus similar to the bovine heart mitochondrial enzyme with respect to prosthetic groups, enzymatic activity, inhibitor sensitivities, and polypeptide subunit composition.     

Purification and characterization of the respiratory nitrate reductase of Bacillus licheniformis.

1. Respiratory nitrate reductase of Bacillus licheniformis was extracted from the bacterial membranes by treatment with deoxycholate and purified to a homogeneous state by means of gel chromatography and anion-exchange chromatography. 2. The enzyme (Mr = 193,000, s20, w = 8.6) consists of two subunits, having apparent molecular weight of 150,000 (alpha subunit) and 57,000 (beta subunit), which are present in an equimolar ratio. It does not contain carbohydrate. Ageing of the enzyme appears to result in splitting of the polypeptide chains at specific sites followed by dissociation and reassociation of the digestion products in various combinations. 3. In contrast to Klebsiella aerogenes repiratory nitrate reductase, which is isolated in a tetrameric form that can be reversibly dissociated into a monomeric form by detergents, B. licheniformis nitrate reductase, after isolation, is always present in a monomeric form. This property is related to the difference in membrane localization of the enzyme in the two organisms. 4. B licheniformis nitrate reductase contains 6.9 atoms of non-heme iron, 6.7 atoms of acid-labile sulfide and 0.93 atoms of molybdenum per molecule of enzyme. The molybdenum seems to be part of a low-molecular weight peptide Mo-cofactor) to which it may be bound by interaction with thiol-groups. 5. Antiserum against the native enzyme contains antibodies against both subunits as well as the Mo-cofactor. The Mo-cofactor does not have any antigenic determininants in common with either the alpha or the beta subunit. Also neither subunit cross-reacts with antiserum against the other subunit. Whereas the respiratory nitrate reductases from K. aerogenes and Escherichia coli are immunologically related, the native enzyme from B. licheniformis does not show any cross-reaction with antiserum prepared against either the K. aerogenes or the E. coli enzyme.     

Purification and further characterization of the second nitrate reductase of Escherichia coli K12

Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli. The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes; it was purified 219 times with a yield of about 11%. It is an Mr-230,000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule. The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum-cofactor-deficient NADPH-dependent nitrate reductase activity from a Neurospora crassa nit-1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme. Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors. Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin. An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z. Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis.     

Biosynthesis of membrane-bound nitrate reductase in Escherichia coli: evidence for a soluble precursor.

Membrane-bound nitrate reductase of Escherichia coli consists of three subunits designated as A, B, and C, with subunit C being the apoprotein of cytochrome b, A hemA mutant that cannot synthesize delta-aminolevulinic acid (ALA) produces a normal, stable, membrane-bound enzyme when grown with ALA. When grown without ALA, this mutant makes a reduced amount of membrane-bound enzyme that is unstable and contains no C subunit. Under the same growth conditions, this mutant accumulates a large amount of a soluble form of the enzyme in the cytoplasm. Accumulation of this cytoplasmic form begins immediately upon induction of the enzyme with nitrate. The cytoplasmic form is very similar to the soluble form of the enzyme obtained by alkaline heat extraction. It is a high-molecular-weight complex with a Strokes radius of 8.0 nm and consists of intact A and B subunits. When ALA is added to a culture growing without ALA, the cytoplasmic form of the enzyme is incorporated into the membrane in a stable form, coincident with the formation of functional cytochrome b. Reconstitution experiments indicate that subunit C is present in cultures grown without ALA but is reduced in amount or unstable. These results indicate that membrane-bound nitrate reductase is synthesized via a soluble precursor containing subunits A and B, which then binds to the membrane upon interaction with the third subunit, cytochrome b.     

Characterization of the respiratory nitrate reductase of Klebsiella aerogenes as a molybdenum-containing iron-sulfur enzyme.

1. In respiratory nitrate reductase I of Klebsiella aerogenes, 0.24 atom of molybdenum, eight iron-sulfur groups and four tightly bound, non-heme iron atoms per molecule of enzyme (Mr 260 000) are found. 2. EPR spectra at 83 degrees K of oxidized and reduced nitrate reductase I show complex lines at g = 2.02 and g = 1.98, which are more intense in the reduced than in the oxidized enzyme. The resonances, the shape and intensity of which are rather temperature insensitive, are attributed to two species of paramagnetic molybdenum. In dithionite-reduced enzyme all these lines are saturated at the same microwave power of 15 mW. This is not the case in oxidized enzyme, where the resonance at g = 2.02 is hard to saturate. Addition of nitrate to dithionite-reduced reductase I decreases the intensity of the EPR lines to about that of oxidized enzyme. The participation of molybdenum in the electron transfer process has been discussed. 3. At 18 degrees K the oxidized enzyme exhibits an axial-symmetrical signal with g parallel = 2.10 and g = 2.03, and a signal with unknown symmetry at g = 2.015. Upon reduction by dithionite, a ferredoxin type of signal is observed with g values at 2.05, 1.95 and 1.88, while the g = 2.015 signal disappears. Reoxidation by nitrate causes a concomitant disappearance of the ferredoxin type of signal and reappearance of the g = 2.015 signal; hence iron-sulfur centres participate in the transfer of electrons to nitrate. 4. Nitrate reductase II, containing only two (Mr 117 000 and 57 000) of the three subunits found in nitrate reductase I and lacking the tightly bound iron, does not exhibit the axial-symmetrical signal (g = 2.10 and 2.03). Thus, it suggested that this signal in nitrate reductase I stems from an iron centre in the low-molecular weight subunit (Mr 52 000). 5. Inhibition studies confirm the participation of metals in the transfer of electrons from reduced benzylviologen to nitrate and show that the binding sites for these substrates are different.     

Anaerobic cytochrome b1 in Escherichia coli: association with and regulation of nitrate reductase.

Nitrate reductase solubilized from the membrane of Escherichia coli by alkaline heat treatment was purified to homogeneity and used to prepare specific antibody. Nitrate reductase, precipitated by this antibody from Triton extracts of the membrane, contained a third subunit, not present in the purified enzyme used to prepare the antibody. This third subunit was identified as the cytochrome b1 apoprotein. This cytochrome is bound to nitrate reductase from wild-type E. coli in a ratio of 2 mol of cytochrome per mol of enzyme complex. In mutants unable to synthesize heme, this cytochrome b1 apoprotein is not bound to nitrate reductase. In these same mutants, the enzyme is overproduced and accumulates in the cytoplasm. The absence of cytochrome also affects the stability of the membrane-bound form of the enzyme.     

Immunological analysis of the heme proteins present in aerobically grown Escherichia coli.

Immunological methods were used to obtain information about Escherichia coli heme proteins. There is a membrane-bound catalase which consists of a single subunit (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis) which is also present in the soluble fraction. Antibodies raised against purified, soluble cytochrome b562 showed that this cytochrome is not related to any of the membrane-bound cytochromes, including the b562 component of the cytochrome o complex. Cytochrome b556 is immunologically unrelated to the cytochrome b556 NR associated with the nitrate reductase system. Cytochrome b556 and cytochrome o are not present in a constant ratio in the membrane.     

The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide     

Resolution of distinct membrane-bound enzymes from Enterobacter cloacae SLD1a-1 that are responsible for selective reduction of nitrate and selenate oxyanions

Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with alpha and beta subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (alphabetagamma) complex with an apparent molecular mass of approximately 600 kDa. The individual subunit sizes are approximately 100 kDa (alpha), approximately 55 kDa (beta), and approximately 36 kDa (gamma), with a predicted overall subunit composition of alpha3beta3gamma3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent Km for selenate was determined to be approximately 2 mM, with an observed Vmax of 500 nmol SeO4(2-) min(-1) mg(-1) (kcat, approximately 5.0 s(-1)). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.     

Reduced nicotinamide adenine dinucleotide-nitrate reductase of Chlorella vulgaris. Purification, prosthetic groups, and molecular properties.

NADH:nitrate reductase (EC 1.6.6.1) from Chlorella vulgaris has been purified 640-fold with an over-all yield of 26% by a combination of protamine sulfate fractionation, ammonium sulfate fractionation, gel chromatography, density gradient centrifugation, and DEAE-chromatography. The purified enzyme is stable for more than 2 months when stored at minus 20 degrees in phosphate buffer (pH 6.9) containing 40% (v/v) glycerol. After the initial steps of the purification, a constant ratio of NADH:nitrate reductase activity to NADH:cytochrome c reductase and reduced methyl viologen:nitrate reductase activities was observed. One band of protein was detected after polyacrylamide gel electrophoresis of the purified enzyme. This band also gave a positive stain for heme, NADH dehydrogenase, and reduced methyl viologen:nitrate reductase. One band, corresponding to a molecular weight of 100, 000, was detected after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme contains FAD, heme, and molybdenum in a 1:1:0.8 ratio. One "cyanide binding site" per molybdenum was found. No non-heme-iron or labile sulfide was detected. From a dry weight determination of the purified enzyme, a minimal molecular weight of 152, 000 per molecule of heme or FAD was calculated. An s20, w of 9.7 S for nitrate reductase was found by the use of sucrose density gradient centrifugation and a Stokes radius of 89 A was estimated by gel filtration techniques. From these values, and the assumption that the partial specific volume is 0.725 cc/g, a molecular weight of 356, 000 was estimated for the native enzyme. These data suggest that the native enzyme contains a minimum of 2 molecules each of FAD, heme, and molybdenum and is composed of at least three subunits.     

Purification of Vibrio fischeri nitrite reductase and its characterization as a hexaheme c-type cytochrome

Dissimilatory nitrite reductase was isolated from anaerobically nitrate-grown Vibrio fischeri cells and purified to electrophoretic homogeneity. The enzyme catalyzes the six-electron reduction of nitrite to ammonia. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under either nonreducing or reducing conditions, the purified nitrite reductase migrated as a single protein band of Mr 57,000. Gel filtration chromatography revealed a native molecular weight of 58,000, indicating the enzyme as isolated to be present in the monomeric form. Purified nitrite reductase exhibited typical c-type cytochrome absorption spectra with the reduced alpha-band at 552.5 nm. Heme content analysis using the purified preparation indicated the enzyme to contain 5.5 heme c groups per molecule. Iron analysis showed the presence of 5.62 g iron atoms per mole of enzyme and no nonheme irons were detected. These results clearly indicate that, similar to the dissimilatory nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes, and Escherichia coli, the V. fischeri nitrite reductase is a hexaheme c-type cytochrome. Amino acid composition of V. fischeri also revealed close similarities to those of the other three hexaheme nitrite reductases previously studied. Based on this information, it is concluded that the four ammonia-forming, dissimilatory nitrite reductases isolated to date represent a homologous group of proteins with the distinct property of being hexaheme c-type cytochromes.     

Limited proteolysis of the nitrate reductase from spinach leaves

The functional structure of assimilatory NADH-nitrate reductase from spinach leaves was studied by limited proteolysis experiments. After incubation of purified nitrate reductase with trypsin, two stable products of 59 and 45 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of 45 kDa was purified by Blue Sepharose chromatography. NADH-ferricyanide reductase and NADH-cytochrome c reductase activities were associated with this 45-kDa fragment which contains FAD, heme, and NADH binding fragment. After incubation of purified nitrate reductase with Staphylococcus aureus V8 protease, two major peaks were observed by high performance liquid chromatography size exclusion gel filtration. FMNH2-nitrate reductase and reduced methyl viologen-nitrate reductase activities were associated with the first peak of 170 kDa which consists of two noncovalently associated (75-90-kDa) fragments. NADH-ferricyanide reductase activity, however, was associated with the second peak which consisted of FAD and NADH binding sites. Incubation of the 45-kDa fragment with S. aureus V8 protease produced two major fragments of 28 and 14 kDa which contained FAD and heme, respectively. These results indicate that the molybdenum, heme, and FAD components of spinach nitrate reductase are contained in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex.     

Purification and properties of dimethylsulfoxide reductase containing a molybdenum cofactor from a photodenitrifier, Rhodopseudomonas sphaeroides f.s. denitrificans

Dimethylsulfoxide (DMSO) reductase was purified to electrophoretic homogeneity from the periplasmic fraction of a photodenitrifier, Rhodopseudomonas sphaeroides f.s. denitrificans. The enzyme had a molecular weight of 82,000 and had no subunit. It contained 1 mol of molybdenum per mol of enzyme, but iron and acid-labile sulfur were not present. The UV-visible spectrum showed only one absorption maximum at 280 nm. Denaturation of the enzyme released a molybdopterin cofactor, the fluorescence spectra of which were almost the same as those of a form B derivative of molybdopterin found in formate dehydrogenase. The Km value for DMSO was 15 microM, which was much lower than that for trimethylamin-N-oxide (TMAO), whereas Vmax with TMAO was larger than that with DMSO.     

Further Characterization of the Reduced Nicotinamide Adenine Dinucleotide Phosphate:Nitrate Oxidoreductase in Aspergillus nidulans

The reduced nicotinamide adenine dinucleotide phosphate (NADPH):nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans wild-type bi-1 was purified by means of salt fractionation, gel filtration, affinity chromatography, and polyacrylamide gel electrophoresis. Enzyme which was adsorbed on Cibacron blue agarose could be eluted with 2 mM NADPH or 2 mM oxidized NADP (NADP(+)), the former being about three times more effective than the latter. About half the total NADPH:nitrate reductase activity adsorbed on agarose required elution with 1 M NaCl. This salt-elutable form remained active with NADPH and was not converted to the NADPH-elutable form after readsorption on Cibacron blue agarose. The NADPH-eluted enzyme exhibited a markedly different electrophoretic mobility than the enzyme eluted with NADP(+) or NaCl. After electrophoresis on polyacrylamide gels, the NADPH-eluted NADPH:nitrate reductase was separated into four proteins, two of which contained nonheme iron and exhibited reduced methyl viologen-nitrate reductase activity. None of these proteins, singly or in combination, reduced nitrate with NADPH as substrate. Difference spectra analyses and specific heme iron stains revealed the presence of cytochrome b(557) in the largest of the proteins. The molecular weights of the four proteins, which were determined from the relationship of their mobilities on varied concentrations of acrylamide gel, were 360,000, 300,000, 240,000, and 118,000. The subunit molecular weights of these, which are determined via sodium dodecyl sulfate slab gel electrophoresis, were 49,000, 50,000, and 75,000. The key role of NADPH in maintenance of the active form of the heteromultimer is further substantiated.     

Synthesis of nitrate reductase components in chlorate-resistant mutants of Escherichia coli.

Specific antibody to purified nitrate reductase from Escherichia coli was used to identify enzyme components present in mutants which lack functional nitrate reductase. chlA and B mutants contained all three subunits present in the wild-type enzyme. Different peptides with a broad range of molecular weights could be precipitated from chlCmutants, and chlE mutants contained either slightly degraded enzyme subunits or no precipitable protein. No mutants produced significant amounts of cytoplasmic enzyme. The chlA and B loci are suggested to function in the synthesis and attachment of a molybdenum-containing factor. The chlC locus is suggested to be the structural gene for nitrate reductase subunit A and chlE is suggested to be involved in the synthesis of the cytochrome b1 apoprotein.