Effects of molybdenum and tungsten on induction of nitrate reductase and formate dehydrogenase in wild type and mutant Paracoccus denitrificans

Molybdenum is required for induction of nitrate reductase and of NAD-linked formate dehydrogenase activities in suspensions of wild type Paracoccus denitrificans; tungsten prevents the development of these enzyme activities. The wild type forms a membrane protein M _r150,000 when incubated with tungsten and inducers of nitrate reductase and this is presumed to represent an inactive form of the enzyme. Suspensions of mutant M-1 did not develop nitrate reductase or formate dehydrogenase activities but the membrane protein M _r150,000 was formed under all conditions tested, including without inducers and without molybdenum. Analysis of membranes, solubilized with deoxycholate, by polyacrylamide gel electrophoresis under nondenaturing conditions showed that the mutant protein had similar electrophoretic mobility to the active nitrate reductase formed by the wilde type. Autoradiography of preparations from cells incubated with ⁵⁵Fe showed that the mutant and wild type proteins contained iron. However, in similar experiments with ⁹⁹Mo, incorporation of molybdenum into the mutant protein was not detectable.We conclude that mutant M-1 is defective in one or more steps required to process molybdenum for incorporation into molybdoenzymes. This failure affects the normal regulation of nitrate reductase protein with respect to the role of inducers.Non-Standard Abbreviations DOC deoxycholate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Analysis of cytoplasmic membrane from wild type and mutant Paracoccus denitrificans containing excess nitrate reductase protein and cytochrome b

Cytoplasmic membranes were isolated from wild type and mutants strain M-1 of Paracoccus denitrificans grown with low aeration to promote synthesis of nitrate reductase protein and cytochrome b. The presence of 10-100-fold excess of nitrate reductase in the wild type or the corresponding enzymically inactive protein in the mutant did not significantly affect respiratory oxidase activities with NADH, succinate or TMPD-ascorbate as electron donor. A cytochrome b-nitrate reductase complex was resolved by isoelectric focussing of Triton X-100 solubilized membranes from the wild type grown with azide and from the mutant, whereas the enzyme complex from nitrate-grown wild type was not resolved from cytochrome c. Preparations from azideinduced wild type or from the mutant could be a suitable source of the cytochrome b associated with nitrate reductase for more detailed studies.Non standard abbreviations IEF isoelectric focussing - TMPD N, N, N, N-tetramethylphenylenediamine - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Evidence for a Second Nitrate Reductase Activity That Is Distinct from the Respiratory Enzyme in Salmonella typhimurium

Significant nitrate reductase activity was detected in mutants of Salmonella typhimurium which mapped at or near chlC and which were incapable of growth with nitrate as electron acceptor. The same mutants were sensitive to chlorate and performed sufficient nitrate reduction to permit anaerobic growth with nitrate as the sole nitrogen source in media containing glucose. The mutant nitrate-reducing protein did not migrate with the wild-type nitrate reductase in polyacrylamide electrophoretic gels. Studies of the electrophoretic mobility in gels of different polyacrylamide concentration revealed that the wild-type and mutant nitrate reductases differed significantly in both size and charge. The second enzyme also differed from the wild-type major enzyme in its response to repression by low pH and its lack of response to repression by glucose. The same mutants were found to be derepressed for nitrite reductase and for a cytochrome with a maximal reduced absorbance at 555 nm at 25°C. This cytochrome was not detected in preparations of the wild type grown under the same conditions. Extracts of these mutants contained normal amounts of the b-type cytochromes which, in the wild type, were associated with nitrate reductase and formate dehydrogenase, respectively, although they could not mediate the oxidation of these cytochromes with nitrate. They were capable of oxidizing the derepressed 555-nm peak cytochrome with nitrate. It is suggested that these mutants synthesize a nitrate-reducing enzyme which is distinct from the chlC gene product and which is repressed in the wild type during anaerobic growth with nitrate.     

Purification of Two Nitrate Reductases from Xanthomonas maltophilia Grown in Aerobic Cultures

Xanthomonas maltophilia ATCC 17666 is an obligate aerobe that accumulates nitrite when grown on nitrate. Spectra of membranes from nitrate-grown cells exhibited b-type cytochrome peaks and A_615-630 indicative of d-type cytochrome but no absorption peaks corresponding to c-type cytochromes. The nitrate reductase (NR) activity was located in the membrane fraction. Triton X-100-extracted reduced methyl viologen-NRs were purified on DE-52, hydroxylapatite, and Sephacryl S-300 columns to specific activities of 52 to 67 μmol of nitrite formed per min per mg of protein. The cytochrome-containing NR_I separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis into a 135-kDa α-subunit, a 64-kDa β-subunit, and a 23-kDa γ-subunit with relative band intensities indicative of a 1:1:1 α/β/γ subunit ratio and a M_r of 222,000. The electronic spectrum of dithionite-reduced purified NR displayed peaks at 425, 528, and 558 nm, indicative of the presence of a cytochrome b, an interpretation consistent with the pyridine hemochrome spectrum formed. The cytochrome b of the NR was reduced under anaerobic conditions by menadiol and oxidized by nitrate with the production of nitrite. This NR contained 0.96 Mo, 12.5 nonheme iron, and 1 heme per 222 kDa: molybdopterin was detected with the Neurospora crassa nit-1 assay. A smaller reduced methyl viologen-NR (169 kDa), present in various concentrations in the Triton X-100 preparations, lacked a cytochrome spectrum and did not oxidize menadiol. The characteristics of the NRs and the absence of c-type cytochromes provide insights into why X. maltophilia accumulates nitrite.     

Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b ₅₅₄ (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b ₅₅₆ (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth. Received: 21 August 1996 / Accepted: 24 October 1996     

A temperature-sensitive mutant of Neurospora crassa deficient in cytochrome b.

Summary A nuclear gene mutant of Neurospora crassa designated cyb-3 is deficient in cytochrome b and coenzyme QH₂-cytochrome c reductase. Nearly normal when grown at 25°C, the strain expresses a mutant phenotype at 38°C. Mitochondria from cybr-3 mycelium, which has undergone 3–4 mass doublings at the elevated temperature, possess 3-fold less cytochrome b, 2-fold more cytochrome, c, 5-fold less coenzyme QH₂-cytochrome c reductase activity, and require 3-fold less antimycin A per milligram of protein to inhibit NADH oxidation than do wild type mitochondria. The activity of coenzyme QH₂-cytochrome c reductase declines rather slowly in cultures of cyb-3 transferred to 38° C, and the in vitro thermostability of the enzyme is very similar in wild type and mutant mitochondria. Therefore, the mutation may decrease synthesis or impair integration into the membrane of cytochrome b and perhaps other proteins of the enzyme complex.Contribution No. 1294-j, Division of Biology, Agricultural Experiment Station, Kansas State University, Manhattan, Kansas.     

Identification of b,c, and d cytochromes in the membrane of Vitreoscilla

Cytochromes b, c, d, and o were identified by spectroscopic analysis of respiratory membrane fragments from Vitreoscilla sp., strain C1. Carbon monoxide difference spectra of the reduced membranes had absorption maxima at 416, 534, and 571 nm (ascribed to cytochrome o) and 632 nm (cytochrome d). Derivative spectra of the pyridine hemochromogen spectra of the membranes identified the presence of b- and c-type cytochromes in Vitreoscilla. The cyanide binding curve of the membranes was biphasic with dissociation constants of 2.14 mM and 10.7 mM which were assigned to cytochrome o and cytochrome d, respectively. Membranes bound carbon monoxide with dissociation constant 3.9 M, which was assigned to cytochrome o. Cytochrome c ₅₅₆ and a NADH-p-iodonitrotetrazolium violet reductase component were partially purified from Vitreoscilla membranes.Abbreviations INT p-iodonitrotetrazolium violet - RMF respiratory membrane fragments - K _d dissociation constant - CHAPS 3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Characterization of subunit structural alterations which occur during purification of nitrate reductase from Escherichia coli

Nitrate reductase from Escherichia coli, purified to homogeneity after release from membranes by deoxycholate treatment, was composed of two subunits of 155,000 (α) and 58,000 (β) daltons and contained no cytochrome b₁. Analysis of fractions at different stages of purification by gel electrophoresis and immunoprecipitation revealed that during the early steps of the purification cytochrome b₁ dissociated from the enzyme and the β subunit was altered in size as determined by sodium dodecyl sulfate-gel electrophoresis. Analysis of the peptide patterns obtained by partial proteolysis of isolated α and β subunits established that these subunits are composed of distinct sequences and ruled out a precursor-product relationship between the two subunits. The β subunit was altered during the purification by loss of a 2000-dalton fragment, apparently from its carboxyl terminus. The protease inhibitor tosyllysine chloromethylketone protected nitrate reductase from more extensive degradation by endogenous proteases during the purification but did not prevent the removal of the 2000-dalton fragment. This carboxyl terminal fragment was part of a 15,000-dalton sequence which was removed by trypsin and which was required for the self-associating character of the unmodified enzyme monomers. From the structural changes which occurred during the purification procedure, it is proposed that the carboxyl terminal segment of the β subunit is involved in the binding of nitrate reductase to cytochrome b₁ and its association with the membrane.     

Membrane-bound nitrite oxidoreductase of Nitrobacter: evidence for a nitrate reductase system

Nitrite oxidoreductase, the essential enzyme complex of nitrite oxidizing membranes, was isolated from cells of the nitrifying bacterium Nitrobacter hamburgensis. The enzyme system was solubilized and purified in the presence of 0.25% sodium deoxycholate. Nitrite oxidoreductase oxidized nitrite to nitrate in the presence of ferricyanide. The pH optimum was 8.0, and the apparent K _m value for nitrite amounted to 3.6 mM. With reduced methyl-and benzylviologen nitrite oxidoreductase exhibited nitrate reductase activity with an apparent K _m value of 0.9 mM for nitrate. NADH was also a suitable electron donor for nitrate reduction. The pH optimum was 7.0.Treatment with SDS resulted in the dissociation into 3 subunits of 116,000, 65,000 and 32,000. The enzyme complex contained iron, molydbenum, sulfur and copper. A c-type cytochrome was present. Isolated nitrite oxidoreductase is a particle of 95±30 Å in diameter.Abbreviation DOC sodium deoxycholate     

In vitro restoration of nitrate reductase: Investigation of Aspergillus nidulans and Neurospora crassa nitrate reductase mutants

Extracts of Aspergillus nidulans wild type (bi-1) and the nitrate reductase mutant niaD-17 were active in the in vitro restoration of NADPH-dependent nitrate reductase when mixed with extracts of Neurospora crassa, nit-1. Among the A. nidulans cnx nitrate reductase mutants tested, only the molybdenum repair mutant, cnxE-14 grown in the presence of 10⁻³ M Na₂MoO₄ was active in the restoration assay.Aspergillus extracts contained an inhibitor(s) which was measured by the decrease in NADPH-dependent nitrate reductase formed when extracts of Rhodospirillum rubrum and N. crassa, nit-1 were incubated at room temperature. The inhibition by extracts of A. nidulans, bi-1, cnxG-4 and cnxH-3 was a linear function of time and a logarithmic function of the protein concentration in the extract.The molybdenum content of N. crassa wild type and nit-1 mycelia were found to be similar, containing approx. 10 μg molybdenum/mg dry mycelium. The NADPH-dependent cytochrome c reductase associated with nitrate reductase was purified from both strains. The enzyme purified from wild-type N. crassa contained more than 1 mol of molybdenum per mol of enzyme, whereas the enzyme purified from nit-1 contained negligible amounts of molybdenum.     

Regulation of reductase formation in Proteus mirabilis

Summary The levels of several redox enzymes in a chlorate-resistant mutant of Proteus mirabilis, which is partially affected in the formation of formate hydrogenlyase, thiosulfate reductase and tetrathionate reductase, were compared with those of the wild type. The composition of the electron transport system of both strains was almost the same in cells grown aerobically, but very different in cells grown anaerobically. In the mutant, the cytochrome content increased twofold, whereas the level of the anaerobic enzymes is strongly diminished. The anaerobic formation of electron transport components in the mutant was, in contrast to that of the wild type, not influenced significantly by azide. During anaerobic growth with nitrate low levels of a functional nitrate reductase system were formed in the mutant. Under these conditions the formation of formate dehydrogenase, formate hydrogenlyase, formate oxidase, thiosulfate reductase, tetrathionate reductase, cytochrome b_563,5 and partly that of cytochrome a₂, was repressed. The repressive effect of nitrate, however, was completely abolished by azide. Therefore, it seems likely that a functional nitrate reductase system, rather than nitrate, controls the formation of the enzymes repressible by nitrate.     

Resolution and reconstitution of succinate-cytochrome c reductase. Preparations and properties of high purity succinate dehydrogenase and ubiquinol—cytochrome c reductase

An improved method was developed to sequentially fractionate succinate-cytochrome c reductase into three reconstitutive active enzyme systems with good yield: pure succinate dehydrogenase, ubiquinone-binding protein fraction and a highly purified ubiquinol-cytochrome c reductase (cytochrome b-c₁ III complex).An extensively dialyzed succinate-cytochrome c reductase was first separated into a succinate dehydrogenase fraction and the cytochrome b-c₁ complex by alkali treatment. The resulting succinate dehydrogenase fraction was further purified to homogeneity by the treatment of butanol, calcium phosphate gel adsorption and ammonium sulfate fractionation under anaerobic condition in the presence of succinate and dithiothreitol. The cytochrome b-c₁ complex was separated into cytochrome b-c₁ III complex and ubiquinone-binding protein fractions by careful ammonium acetate fractionation in the presence of deoxycholate.The purified succinate dehydrogenase contained only two polypeptides with molecular weights of 70 000 and 27 000 as revealed by the sodium dodecyl sulfate polyacrylamide gel electrophoretic pattern. The enzyme has the reconstitutive activity and a low K_m ferricyanide reductase activity of 85 μmol succinate oxidized per min per mg protein at 38°C.Chemical composition analysis of cytochrome b-c₁ III complex showed that the preparation was completely free of contamination of succinate dehydrogenase and ubiquinone-binding protein and was 30% more pure than the available preparation.When these three components were mixed in a proper ratio, a thenoyl-trifluoroacetone- and antimycin A-sensitive succinate-cytochrome c reductase was reconstituted.     

The circadian rhythm in Bryophyllum leaves: Phase control by radiant energy

An inactivated nitrate reductase (EC 1.6.6.1) formed in vivo by the green alga Chlorella fusca Shihira and Kraus is shown to be a cyanide complex. The partially purified inactive enzyme releases 0.048 nmol of HCN per unit of enzyme activated. This compares with 0.066 nmol of HCN liberated in similar previous measurements with the inactivated enzyme from Chlorella vulgaris. The nitrate reductase from C. fusca has been purified to a level of 67 mol nitrate reduced per min per mg enzyme. It contains a cytochrome b₅₅₇, at a level 1.9-fold higher per unit of active enzyme, than the nitrate reductase from C. vulgaris.Abbreviations FAD flavin-adenine dinucleotide - NADH nicotineamide-adenine-dinucleotide (reduced)     

The correlation between the protein composition of cytoplasmic membranes and the formation of nitrate reductase A,chlorate reductase C and tetrathionate reductase in Proteus mirabilis wild type and some chlorate resistant mutants

Three genotypically different chlorate resistant mutants, chl I, chl II and chl III, appeared to lack completely nitrate reductase A, chlorate reductase C and tetrathionate reductase activity. Fumarate reductase is only partially affected in chl I and chl III and unaffected in chl II. Formate dehydrogenase is only partially diminished in chl II, hydrogenase is diminished in chl I and chl II and completely absent in chl III.Subunits of nitrate reductase A, chlorate reductase C and tetrathionate reductase have been identified in protein profiles of purified cytoplasmic membranes from the wild type and the three mutant strains, grown under various conditions. Only the presence and absence of the largest subunits of these enzymes appeared to be correlated with their repression and derepression in the wild type membranes. On the cytoplasmic membranes of the chl I and chl III mutants these subunits lack for the greater part. In the chl II mutant, however, these subunits are inserted in the membrane all together after anaerobic growth with or without nitrate.A model for the repression/derepression mechanism for the reductases has been proposed. It includes repression by cytochrome b components, whereas the redox-state of the nitrate reductase A molecule itself is also involved in its derepression under anaerobic conditions.     

Mode of action of natural inactivator proteins from corn and rice on a purified assimilatory nitrate reductase

The molecular basis for the action of two natural inactivator proteins, isolated from rice and corn, on a purified assimilatory nitrate reductase has been examined by several physical techniques. Incubation of purified Chlorella nitrate reductase with either rice inactivator protein or corn inactivator protein results in a loss of NADH:nitrate reductase and the associated partial activity, NADH:cytochrome c reductase, but no loss in nitrate-reducing activity with reduced methyl viologen as the electron donor. The molecular weight of the reduced methyl viologen:nitrate reductase species, determined by sedimentation equilibrium in the Beckman airfuge after complete inactivation with rice inactivator protein or with corn inactivator protein, was 595,000 and 283,000, respectively, compared to a molecular weight of 376,000 for the untreated control determined under the same conditions. Two protein peaks were observed after molecular-sieve chromatography on Sephacryl S-300 of nitrate reductase inactivated by corn inactivator protein. The Stokes radii of these fragments were 68 and 24 Å, compared to a value of 81 Å for untreated nitrate reductase. The large fragment contained molybdenum and heme but no flavin, and had nitrate-reducing activity with reduced methyl viologen as electron donor. The small fragment contained FAD but had no NADH:cytochrome c reductase or nitrate-reducing activities. Molecular weights determined by sodium dodecyl sulfate-gel electrophoresis were 67,000 and 28,000 for the large and small fragments, respectively, compared to a subunit molecular weight of 99,000 determined for the untreated control. No change in subunit molecular weight of nitrate reductase after inactivation by rice inactivator protein was observed. These results indicate that rice inactivator protein acts by binding to nitrate reductase. The stoichiometry of binding is 1–2 molecules of rice inactivator protein to one tetrameric molecule of nitrate reductase. Corn inactivator protein, in contrast, acts by cleavage of a M_r 30,000 fragment from nitrate reductase which is associated with FAD. The remaining fragment is a tetramer of M_r 70,000 subunits which retains nitrate-reducing activity and contains molybdenum and heme but has no NADH:dehydrogenase activity. The action of rice inactivator protein was partially prevented by NADH and completely prevented by a combination of NADH and cyanide, while the action of corn inactivator protein was not significantly affected by these effectors.     

The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b(5)

Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b₅. In both instances, product formation occurs. When the second electron is donated by cytochrome b₅, catalysis (product formation) is ∼10- to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼15% the coupling of NADPH consumption to product formation. Cytochrome b₅ has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b₅ on cytochrome P450 2B4 reactivity can explain how cytochrome b₅ is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b₅ to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochrome b₅ to cytochrome P450 reductase, cytochrome b₅ inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b₅ are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b₅ stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase.     

Isolation and nucleotide sequence of the hmp gene that encodes a haemoglobin-like protein in Escherichia coli K-12

Summary In the course of an attempt to identify genes that encode Escherichia coli dihydropteridine reductase (DHPR) activities, a chromosomal DNA fragment that directs synthesis of two soluble polypeptides of M_r 44000 and 46000 was isolated. These proteins were partially purified and were identified by determination of their N-terminal amino acid sequences. The larger was serine hydroxymethyltransferase, encoded by the glyA gene, while the smaller was the previously described product of an unnamed gene closely linked to glyA, and transcribed in the opposite direction. Soluble extracts of E. coli cells that overproduced the 44 kDa protein had elevated DHPR activity, and were yellow in colour. Their visible absorption spectra were indicative of a CO-binding b-type haemoprotein that is high-spin in the reduced state. The sequence of the N-terminal 139 residues of the protein, deduced from the complete nucleotide sequence of the gene, had extensive homology to almost all of Vitreoscilla haemoglobin. We conclude that E. coli produces a soluble haemoglobin-like protein, the product of the hmp gene (for haemoprotein). Although the protein has DHPR activity, it is distinct from the previously purified E. coli DHPR.     

Defects in cytochrome cd 1-dependent nitrite respiration of transposon Tn5-induced mutants from Pseudomonas stutzeri

Mutants with defective respiratory nitrite utilization (Nir^- phenotype) were obtained by transposon Tn5 insertion into genomic DNA of the ZoBell strain of Pseudomonas stutzeri. Three representative mutants were characterized with respect to their activities of nitrite and nitric oxide reduction, cytochrome cd ₁ content, and pattern of soluble c-type cytochromes. Mutant strain MK201 over-produced cytochrome c ₅₅₂ about fourfold by comparison with the wild type, but possessed an in vitro functional cytochrome cd ₁. Mutant strain MK202 lacked cytochrome cd ₁ and, simultaneously, had low amounts of cytochrome c ₅₅₂ and the split -peak c-type cytochrome. Strain MK203 synthesized nitrite reductase defective in the heme d ₁ prosthetic group. Irrespective of these biochemically distinct Nir^- phenotypes, all mutants preserved the nitric oxidereducing capability of the wild type. The mutant characteristics demonstrate that cytochrome cd ₁ is essential for nitrite respiration of P. stutzeri and establish the presence of a nitric oxide-reducing system distinct from cytochrome cd ₁. They also indicate the functional or regulatory interdependence of c-type cytochromes.