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Nitrate Reductase from a Neurospora Mutant and a Component of Molybdenum-Enzymes (Nitrogenases/Sulfite Oxidase/E

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Nitrate Reductase from a Neurospora Mutant and a Component of Molybdenum-Enzymes (Nitrogenases/Sulfite Oxidase/E Proc. Nat. Acad. Sci. USA Vol. 68, No. 12, pp. 3242-3246, December 1971 In Vitro Formation of Assimilatory Reduced Nicotinamide Adenine Dinucleotide Phosphate: Nitrate Reductase from a Neurospora Mutant and a Component of Molybdenum-Enzymes (nitrogenases/sulfite oxidase/E. coli) ALVIN NASON, KUO-YUNG LEE, SU-SHU PAN, PAUL A. KETCHUM*, ANTONIO LAMBERTI, AND JAMES DEVRIES McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218. Communicated by Earl R. Stadttman, September 16, 19711 ABSTRACT An active Neurospora-like assimilatory denum moiety. The second component could also be NADPH-nitrate reductase (EC 1.6.6.2), which can be formed in vitro by incubation of extracts of nitrate-in- supplied by the individual molybdenum-enzymes bovine duced Neurospora crassa mutant nit-i with extracts of milk and intestinal xanthine oxidases, chicken liver dehydro- (a) certain other nonallelic nitrate reductase mutants, (b) genase, and rabbit liver aldehyde oxidase (8), provided they uninduced wild type, or (c) xanthine oxidizing and liver were subjected to prior acidification, a treatment known to aldehyde-oxidase systems was also formed by combination dissociate some proteins into their subunits (9-11). (How- of the nit-i extract with other acid-treated enzymes known to contain molybdenum. These molybdenum ever, sodium molybdate and some 20 different partially puri- enzymes included (a) nitrogenase, or its molybdenum- fied enzymes that do not contain molybdenum were inactive.) iron protein, from Clostridium, Azotobacter, and soybeats- In all instances, in vitro formation of enzyme activity was nodule bacteroids, (b) bovine liver sulfite oxidase, (c) accompanied by the conversion of cytochrome c reductase respiratory formate-nitrate reductase from Escherichia = to the faster form coli, (d) NADH-nitrate reductase from foxtail grass (Setaria activity (S20, 4.5S) sedimenting faberii), and (e) FADH2- and reduced methyl viologen- (Seo,,0 = 7.9 S) which is associated with NADPH-nitrate nitrate reductase preparations from certain Neurospora reductase (suggestive of subunit assembly) and the appear- mutants. Several molybdenum-amino-acid complexes, as ance of NADPH-, FADH2-, and MVH-nitrate reductase possible catalytic models of nitrogenase, were inactive (as activities. The assimilatory NADPH-nitrate reductase thus were some previously tested 20 nonmolybdenum enzymes) in place of the acid-treated molybdenum-containing formed is similar to the wild-type Neurospora enzyme. More- enzymes. The results imply the existence of a molybde- over, nit-i is the only one of the nitrate reductase Neurospora num-contain-ing component shared by the known molyb- mutants that also lacks xanthine dehydrogenase, and is denum-enzy n-es. unable to grow on hypoxanthine or nitrate as the sole nitrogen source (8), similar to certain Aspergillus mutants (12). Assimiiilatory NADPH-nitrate reductase (NADPH: nitrate In the present pl)aper, we report that other acid-treated oxidoreductase, EC 1.6.6.2) from Neurospora crassa is an in- mnolybdenum-enzymes from diverse phylogenetic sources can ducible, soluble, cytochrome b-containing, sulfhydryl molyb- also interact with the induced Neurospora nit-i extract to doflavoprotein of molecular weight 230,000. It also possesses form a Neurospora-like assimilatory NADPH-nitrate reduc- inducible NADPH-cytochrome c reductase and reduced tase. The results suggest the existence of a molybdenum-con- flavin-adenine dinucleotide (FADH2)- and reduced methyl taining component, perhaps in the form of a molybdenum viologen (MIVH)-nitrate reductase activities in a constant cofactor, that is shared in common by the known molyb- proportion (1-6). Incubation of a cell-free extract of the denum-enzymes among a diversity of organisms including Neurospora nitrate reductaseless mutant, nit-1, that is in- microbes, plants, and animals. A preliminary account of a duced by nitrate, with extracts of other nitrate reductase portion of this work has been (13). Neurospora mutants, that are nonallelic, or with uninduced published wild type produced the active enzyme, presumably by a com- MATERIALS AND METHODS bination of at least two dissimilar protein subunits, coded for by different cistrons (7). One of the subunits was believed N. crassa wild type and several nitrate reductase mutant to be a nitrate-inducible component(s) of nit-i that is re- strains including nit-i were maintained, grown, and induced sponsible at least for the early part of the electron transport with nitrate and crude extracts prepared as in an earlier sequence, as reflected by its inducible NADPH-cytochrome study (7), unless otherwise noted. Sources of substrates, c reductase activity which is flavin-dependent. The second cofactors and various chemicals have been cited (7). component was inferred to be a constitutive entity that is Sulfite oxidase (EC 1.8.3.1) was purified from beef liver absent in nit-i, but present in all other mutants and according to the procedure of MacLeod et al. (14). uninduced wild type, and is responsible for the latter part Cell-free, high-speed supernatant and pellet fractions of of the nitrate reductase pathway that includes the molyb- Escherichia coli respiratory nitrate reductase (designated S144 and P,44, respectively) that possesses formate-, and to a lesser Abbreviations: FADHS, reduced Flavin-adenine dinucleotide; extent, NADH-nitrate reductase activities were prepared by MVH, reduced methyl viologen. suspension of frozen E. coli K235 cells (15), kindly provided * Present address: Biology Department. Oakland University, by Dr. S. Roseman, in an equal weight of cold 0.1 M phos- Rochester, Mich. phate buffer (pH 7.3)-i mM 2-mercaptoethanol-500 uiM 3242 Downloaded by guest on September 27, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) NADPH-Nitrate Reductase from a Neurospora Mutant 3243 TABLE 1. In vitro formation of assimilatory EDTA ("preparation buffer"), rupture in a French pressure NADPH-nitrate reductase by incubation of individual cell at 10,000 psi and centrifugation at 10,000 X g and acid-treated molybdenum-enzymes with an extract of 144,000 X g for 10 and 90 respectively. The supernatant nitrate-induced Neurospora mutant nit-i min, solution (S144) was used as such, whereas the pellet was washed three times by successive resuspension and centrifu- NADP - NOs- activity formed (pH before incubation with gation before final suspension in preparation buffer (about Mo-enzymes nit-i) one-tenth of the original volume used for rupturing the cells) and components 7.3 2.5 2.0 1.8 to constitute the P,44 fraction. Partially purified preparations Nitrogenases (nmol N02- produced/O.20 ml/60 of assimilatory NADH-nitrate reductase from foxtail grass Expt. 1 min) (Setaria faberii) and the multi-protein complex nitrogenase, (Az. vinelandii) including its two separate components (the molybdenum- Nitrogenase 4.2 4.7 54.4 iron protein and the iron protein) from Azotobacter vinelandii Mo-Fe protein 0 1.0 1.6 7.0 (16, 17), Clostridium pasteurianum (18), and soybean (Glycine Fe protein 0 0 0 max.) nodule bacteroids (19), were kindly provided by the Expt. 2 indicated sources. Acid treatment of proteins and other sub- (Az. vinelandii) stances was usually achieved by a 5- to Nitrogenase 0 7.4 50-fold dilution at Mo-Fe protein 0 14.0 00C in 0.1 N NaCl adjusted with 1 N HOl to the desired Expt. 3 pH. In some instances, more concentrated HOC solutions (Cl. pasteurianum) were used to minimize dilution. After 1-7 min, the acid- Mo-Fe protein 12.6* 11.6 treated preparations were incubated with an equal volume Fe protein 0. 1* - 0.1 of a crude extract of nitrate-induced nit-i (final pH of mixture Expt. 4 6.8) for 30-60 min at room temperature. The NADPH- (soybean nodule bacteroids) nitrate reductase activity thus formed was assayed as de- Nitrogenase 4.0 61.7 scribed (5), generally for 30-60 min, and is expressed in Mo-Fe protein 4.8 - 62.4 nmoles of nitrite produced per assay period. FADHr- and (pH (pH (pH (pH MVH-nitrate reductase activities were determined by the 7.0) 2.4) 2.1) 1.4) (per 10 min assay) usual procedure (6), and formate- and NADH-nitrate Bovine liver reductase activities were determined by the same method as S02- oxidase 0 5.3 83.5 26.1 NADPH-nitrate reductase, except that 0.05 ml of 10 mM sodium formate and NADH, respectively, were used in place The Az. vinelandii nitrogenase and its two metallo-protein of NADPH. All assays were performed with two or more fractions in Expt. 1 were kindly given by Drs. R. C. Bums and different aliquots and were directly proportional to enzyme R. F. W. Hardy, and in Expt. 2 by Dr. W. A. Bulen. In Expt. 1, concentration. Procedures for sucrose gradient sedimentation the nitrogenase fraction contained 35-40 mg of protein/ml, the analysis (SW 39L rotor, 39,000 rpm, for about 20 hr), protein Mo-Fe-protein fraction contained 35 mg protein/m', and the Fe- determination, estimation of Stokes radius by Sephadex protein fraction contained 35 mg of protein/ml. All fractions were G-200 gel filtration, and molecular weight calculation were diluted 1:25 with 0.1 M NaCl solution at the indicated pH the same as (acidified with HC1), maintained thus for 7 min at 0°C, mixed detailed elsewhere (6-8). with an equal volume of crude extract of induced nit-i (about RESULTS 10 mg protein/ml), and the resulting mixture was incubated at room temperature for 30 min. The NADPH-nitrate reductase The earlier findings (7) that the acid-treated xanthine formed was determined by a 60-min. assay. In Expt. 2, the oxidizing and liver aldehyde oxidase systems can interact nitrogenase fraction [in 25 mM Tris HCl (pH 7.5)-0.1 mg di- with an extract of nitrate-induced nit-i to form assimilatory thiothreitol/ml] contained 34.3 mg of protein/ml, and the Mo- NADPH-nitrate reductase prompted a similar examination Fe-protein (in 0.27 M NaCl-25 mM Tris-HCl (pH 7.4)-0.1 mg of other known molybdenum-enzymes.
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