A reduced pyridine nucleotides-diaphorase activity associated to the assimilatory nitrite reductase complex from Neurospora crassa

The Neurospora crassa assimilatory NAD(P)H-nitrite reductase complex has associated a NAD(P)H-diaphorase activity. 1. This NAD(P)H-diaphorase activity can use either mammalian cytochrome c, 2,6--dichlorophenol-indophenol, ferricyanide, or menadione as electron acceptor from the reduced pyridine nucleotides, and requires flavin adenine dinucleotide for maximal activity. 2. It is inhibited by p-hydroxymercuribenzoate, 1 muM, and it is unaffected by cyanide, sulfite, or arsenite at concentrations which completely inhibit the NAD(P)H-nitrite reductase activity. 3. Flavin adenine dinucleotide specifically protects the NAD(P)H-diaphorase activities, but not the NAD(P)H-nitrite reductase activities, against thermal inactivation. 4. In vitro preincubation of the Neurospora crassa nitrite reductase complex with reduced pyridine nucleotides plus flavin adenine dinucleotide inactivates the NAD(P)H-nitrite reductase activities, but does not affect the NAD(P)H-diaphorase activities, indicating that this nitrite reductase inactivation occurs in the part of the enzyme that contain the nitrite reducing center.     

Reactions of the Neurospora crassa nitrate reductase with NAD(P) analogs.

The assimilatory NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) from Neurospora crassa is competitively inhibited by 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP) which are structural analogs of NAD and NADP, respectively. The amino group of the pyridine ring of AAD(P) can react with nitrous acid to yield the diazonium derivative which may covalently bind at the NAD(P) site. As a result of covalent attachment, diazotized AAD(P) causes time-dependent irreversible inactivation of nitrate reductase. However, only the NADPH-dependent activities of the nitrate reductase, i.e. the overall NADPH-nitrate reductase and the NADPH-cytochrome c reductase activities, are inactivated. The reduced methyl viologen- and reduced FAD-nitrate reductase activities which do not utilize NADPH are not inhibited. This inactivation by diazotized AADP is prevented by 1 mM NADP. The inclusion of 1 muM FAD can also prevent inactivation, but the FAD effect differs from the NADP protection in that even after removal of the exogenous FAD by extensive dialysis or Sephadex G-25 filtration chromatography, the enzyme is still protected against inactivation. The FAD-generated protected form of nitrate reductase could again be inactivated if the enzyme was treated with NADPH, dialyzed to remove the NADPH, and then exposed to diazotized AADP. When NADP was substituted for NADPH in this experiment, the enzyme remained in the FAD-protected state. Difference spectra of the inactivated nitrate reductase demonstrated the presence of bound AADP, and titration of the sulfhydryl groups of the inactivated enzyme revealed that a loss of accessible sulfhydryls had occurred. The hypothesis generated by these experiments is that diazotized AADP binds at the NADPH site on nitrate reductase and reacts with a functional sulfhydryl at the site. FAD protects the enzyme against inactivation by modifying the sulfhydryl. Since NADPH reverses this protection, it appears the modifications occurring are oxidation-reduction reactions. On the basis of these results, the physiological electron flow in the nitrate reductase is postulated to be from NADPH via sulfhydryls to FAD and then the remainder of the electron carriers as follows: NADPH leads to -SH leads to FAD leads to cytochrome b-557 leads to Mo leads to NO-3.     

Neurospora crassa NAD(P)H-nitrite reductase. Studies on its composition and structure

Neurospora crassa nitrite reductase (Mr = 290,000) catalyzes the NAD(P)H-dependent 6-electron reduction of nitrite to ammonia via flavin and siroheme prosthetic groups. Homogeneous N. crassa nitrite reductase has been prepared employing conventional purification methods followed by affinity chromatography on blue dextran-Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of homogeneous nitrite reductase reveals a single subunit band of Mr = 140,000. Isoelectric focusing of dissociated enzyme followed by sodium dodecyl sulfate-gel electrophoresis in the second dimension yields a single subunit spot with an isoelectric point at pH 6.8-6.9. Two-dimensional thin layer chromatography of acid-hydrolyzed nitrite reductase treated with 5-dimethylaminoaphthalene-1-sulfonyl chloride yields a single reactive NH2-terminal corresponding to glycine. An investigation of the prosthetic groups of nitrite reductase reveals little or no flavin associated with the purified protein, although exogenously added FAD is required for activity in vitro. An iron content of 9-10 Fe eq/mol suggests the presence of nonheme iron in addition to the siroheme moieties. Amino acid analysis yields 43 cysteinyl residues and sulfhydryl reagents react with 50 thiol eq/mol of nitrite reductase. The non-cysteinyl sulfur content, determined as 8.1 acid-labile sulfide eq/mol, is presumably associated with nonheme iron to form iron-sulfur centers. We conclude that N. crassa nitrite reductase is a homodimer of large molecular weight subunits housing an electron transfer complex of FAD, iron-sulfur centers, and siroheme to mediate the reduced pyridine nucleotide-dependent reduction of nitrite to ammonia.     

Assimilatory nitrate reductase from Acinetobacter calcoaceticus

A soluble nitrate reductase from the bacterium Acinetobacter calcoaceticus grown on nitrate has been characterized. The reduction of nitrate to nitrite is mediated by an enzyme of 96000 molecular weight that can use as electron donors either viologen dyes chemically reduced with dithionite or enzymatically reduced with NAD(P)H, through specific diaphorases which utilize viologens as electron acceptors. Nitrate reductase activity is molybdenum-dependent as shown by tungstate antagonistic experiments and is sensitive to -SH reagents and metal chelators such as KCN.The enzyme synthesis is repressed by ammonia. Moreover, nitrate reductase activity undergoes a quick inactivation either by dithionite and temperature or by dithionite in the presence of small amounts of nitrate. Cyanate prevents this inactivating process and can restore the activity once the inactivation had occurred, thus suggesting that an interconversion mechanism may participate in the regulation of Acinetobacter nitrate reductase.Abbreviations EDTA ethylenediaminetetraacetate - BV benzyl viologen - MV methyl viologen - MW molecular weight - NEM N-ethylmaleimide - p-HMB p-hydroxymercuribenzoate - DCPIP 2,6-dichlorophenol-indophenol - FMN flavin mononucleotide - FAD flavin adenine dinucleotide - KCNO potassium cyanate     

Reduced nicotinamide–adenine dinucleotide–nitrite reductase from Azotobacter chroococcum

1. The assimilatory nitrite reductase of the N(2)-fixing bacterium Azotobacter chroococcum was prepared in a soluble form from cells grown aerobically with nitrate as the nitrogen source, and some of its properties have been studied. 2. The enzyme is a FAD-dependent metalloprotein (mol.wt. about 67000), which stoicheiometrically catalyses the direct reduction of nitrite to NH(3) with NADH as the electron donor. 3. NADH-nitrite reductase can exist in two either active or inactive interconvertible forms. Inactivation in vitro can be achieved by preincubation with NADH. Nitrite can specifically protect the enzyme against this inactivation and reverse the process once it has occurred. 4. A. chroococcum nitrite reductase is an adaptive enzyme whose formation depends on the presence of either nitrate or nitrite in the nutrient solution. 5. Tungstate inhibits growth of the microorganism very efficiently, by competition with molybdate, when nitrate is the nitrogen source, but does not interfere when nitrite or NH(3) is substituted for nitrate. The addition of tungstate to the culture media results in the loss of nitrate reductase activity but does not affect nitrite reductase.     

Identification and characterization of a novel ferric reductase from the hyperthermophilic Archaeon Archaeoglobus fulgidus

Archaeoglobus fulgidus, a hyperthermophilic sulfate-reducing Archaeon, contains high Fe(3+)-EDTA reductase activity in its soluble protein fraction. The corresponding enzyme, which constitutes about 0.75% of the soluble protein, was purified 175-fold to homogeneity. Based on SDS-polyacrylamide gel electrophoresis, the ferric reductase consists of a single subunit with a M(r) of 18,000. The M(r) of the native enzyme was determined by size exclusion chromatography to be 40,000 suggesting that the native ferric reductase is a homodimer. The enzyme uses both NADH and NADPH as electron donors to reduce Fe(3+)-EDTA. Other Fe(3+) complexes and dichlorophenolindophenol serve as alternative electron acceptors, but uncomplexed Fe(3+) is not utilized. The purified enzyme strictly requires FMN or FAD as a catalytic intermediate for Fe(3+) reduction. Ferric reductase also reduces FMN and FAD, but not riboflavin, with NAD(P)H which classifies the enzyme as a NAD(P)H:flavin oxidoreductase. The enzyme exhibits a temperature optimum of 88 degrees C. When incubated at 85 degrees C, the enzyme activity half-life was 2 h. N-terminal sequence analysis of the purified ferric reductase resulted in the identification of the hypothetical gene, AF0830, of the A. fulgidus genomic sequence. The A. fulgidus ferric reductase shares amino acid sequence similarity with a family of NAD(P)H:FMN oxidoreductases but not with any ferric reductases suggesting that the A. fulgidus ferric reductase is a novel enzyme.     

Effects of a nitrate reductase inactivating enzyme and NAD(P)H on the nitrate reductase from higher plants and Neurospora.

Evidence is presented which suggests that the NAD(P)H-cytochrome c reductase component of nitrate reductase is the main site of action of the inactivating enzyme. When tested on the nitrate reductase (NADH) from the maize root and scutella, the NADH-cytochrome c reductase was inactivated at a greater rate than was the FADH2-nitrate reductase component. With the Neurospora nitrate reductase (NADPH) only the NADPH-cytochrome c reductase was inactivated. p-Chloromercuribenzoate at 50 muM, which gave almost complete inhibition of the NADH-cytochrome c reductase fraction of the maize nitrate reductase, had no marked effect on the action of the inactivating enzyme. A reversible inactivation of the maize nitrate reductase has been shown to occur during incubation with NAD(P)H. In contrast to the action of the inactivating enzyme, it is the FADH2-nitrate reductase alone which is inactivated. No inactivation of the Neurospora nitrate reductase was produced by NAD(P)H alone and also in the presence of FAD. The lack of effect of the inactivating enzyme and NAD(P)H on the FADH2-nitrate reductase of Neurospora suggests some differences in its structure or conformation from that of the maize enzyme. A low level of cyanide (0.4 mu M) markedly enhanced the action of NAD(P)H on the maize enzyme; Cyanide at a higher level (6 mu M) did give inactivation of the Neurospora nitrate reductase in the presence of NADPH and FAD. The maize nitrate reductase, when partially inactivated by NADH and cyanide, was not altered as a substrate for the inactivating enzyme. The maize root inactivating enzyme was also shown to inactivate the nitrate reductase (NADH) in the pea leaf. It had no effect on the nitrate reductase from either Pseudomonas denitrificans or Nitrobacter agilis.     

NAD (P) H-dependent reduction of nicotinamide N-oxide by an unique enzyme system consisting of liver microsomal NADPH-cytochrome C reductase and cytosolic aldehyde oxidase

NAD (P) H-dependent reduction of nicotinamide N-oxide was investigated with rabbit liver preparations. Microsomes, microsomal NADPH-cytochrome c reductase or cytosolic aldehyde oxidase alone exhibited no nicotinamide N-oxide reductase activity in the presence of NADPH or NADH. However, when the microsomal preparations were combined with the cytosolic enzyme, a significant N-oxide reductase activity was observed in the presence of the reduced pyridine nucleotide. The activity was enhanced by FAD or methyl viologen. Cytosol alone supplemented with NADPH or NADH exhibited only a slight, but when combined with microsomes, a significant N-oxide reductase activity. Based on these facts, we propose a new electron transfer system consisting of NADPH-cytochrome c reductase and aldehyde oxidase, which exhibits nicotinamide N-oxide reductase activity in the presence of the reduced pyridine nucleotide.     

Putidaredoxin reductase,a new function for an old protein

Properties of recombinant wild type (WT) and six-histidine tag-fused (His(6)) putidaredoxin reductase (Pdr), a FAD-containing component of the soluble cytochrome P450cam monooxygenase system from Pseudomonas putida, have been studied. Both WT and His(6) Pdr were found to undergo a monomer-dimer association-dissociation and were partially present as an NAD(+)-bound form. Although molecular, spectral, and electron transferring properties of recombinant His(6) Pdr to artificial and native electron acceptors were similar to those of the WT protein, the presence of eight additional C-terminal amino acid residues, Pro-Arg-His-His-His-His-His-His, had a crucial effect on the enzyme interaction with oxidized pyridine nucleotide. Under anaerobic conditions, NAD(+) induced in His(6) Pdr spectral changes indicative of flavin reduction and formation of the charge transfer complex between the reduced FAD and NAD(+). The reaction proceeded considerably faster in the presence of free histidine and thiol-reducing agents, such as dithiothreitol and reduced glutathione. In the presence of any of these three reagents, NAD(+) was capable of inducing reduction of the flavin in WT Pdr. Free thiol groups were identified as an internal source of electrons in the enzyme. The results showed that WT and His(6) Pdr were able to function as NAD(H)-dependent dithiol/disulfide oxidoreductases catalyzing both forward and reverse reactions, NAD(+)-dependent oxidation of thiols, and NADH-dependent reduction of disulfides. This function of the flavoprotein can be dissociated from electron transfer to putidaredoxin. Similarity of Pdr to the enzymes of the glutathione reductase family is discussed.     

The NADH dehydrogenase of the respiratory chain of Escherichia coli. II. Kinetics of the purified enzyme and the effects of antibodies elicited against it on membrane-bound and free enzyme.

The purified respiratory chain NADH dehydrogenase of Escherichia coli oxidizes NADH with either dichlorophenolindophenol (DCIP). ferricyanide, or menadione as electron acceptors, with values for NADH are similar with the three electron acceptors (approximately 50 muM). The purified enzyme contains no flavin and has an absolute requirement for FAD, with Km values around 4 muM. The pH optimum of the enzyme appears to be between 6.5 and 7; the optimum is difficult to establish because of nonenzymatic reduction of DCIP at the lower pH values. Potassium cyanide stimulates the DCIP reductase activity about 2-fold, but has no effect on ferricyanide reductase. The enzyme exhibits hyperbolic kinetics with respect to NADH concentration in both the ferricyanide and DCIP reductase assays, but cooperatively is seen in the menadione reductase reaction. NAD+ is an effective competitive inhibitor of the reaction (Ki congruent to 20 muM); in the presence of NAD+, the NADH saturation curve becomes cooperative, even in the DCIP reductase assay. Many adenine containing nucleotides are competitive inhibitors of the enzyme. The apparent Ki values for these nucleotides as inhibitors of the purified enzyme, the membrane-bound NADH dehydrogenase, and the NADH oxidase are equivalent. An examination of inhibitory effects of a series of adenine nucleotides suggests that the inhibitors act as analogues of NAD+, which is the true physiological inhibitor. The results suggest that the enzyme in situ is always partially inhibited by the levels of NAD- in the E coli cell, and thus behaves in a cooperative fashion to changes in the NAD+/NADH ratio. An antibody has been elicited against the purified NADH dehydrogenase. Immunodiffusion and crossed immunoelectrophoresis show that the antibody is directed principally against the NADH dehydrogenase, with some activity against minor contaminants in the purified preparation. The antibody inhibits NADH dehydrogenase activity 50% at saturating levels. When this antibody preparation is used to examine solubilized membrane preparations, two major immunoprecipitates are found. A parallel inhibition of the membrane-bound NADH dehydrogenase and NADH oxidase activities is seen, supporting the hypothesis that the purified enzyme is indeed a component of the respiratory chain-dependent NADH oxidase pathway.     

Monodehydroascorbate reductase from cucumber is a flavin adenine dinucleotide enzyme

Monodehydroascorbate reductase (EC 1.6.5.4) was purified from cucumber fruit to a homogeneous state as judged by polyacrylamide gel electrophoresis. The cucumber monodehydroascorbate reductase was a monomer with a molecular weight of 47,000. It contained 1 mol of FAD/mol of enzyme which was reduced by NAD(P)H and reoxidized by monodehydroascorbate. The enzyme had an exposed thiol group whose blockage with thiol reagents inhibited the electron transfer from NAD(P)H to the enzyme FAD. Both NADH and NADPH served as electron donors with Km values of 4.6 and 23 microM, respectively, and Vmax of 200 mol of NADH and 150 mol of NADPH oxidized mol of enzyme-1 s-1. The Km for monodehydroascorbate was 1.4 microM. The amino acid composition of the enzyme is presented. In addition to monodehydroascorbate, the enzyme catalyzed the reduction of ferricyanide and 2,6-dichloroindophenol but showed little reactivity with calf liver cytochrome b5 and horse heart cytochrome c. The kinetic data suggested a ping-pong mechanism for the monodehydroascorbate reductase-catalyzed reaction. Cucumber monodehydroascorbate reductase occurs in soluble form and can be distinguished from NADPH dehydrogenase, NADH dehydrogenase, DT diaphorase, microsome-bound NADH-cytochrome b5 reductase, and NADPH-cytochrome c reductase by its molecular weight, amino acid composition, and specificity of electron acceptors and donors.     

Variations in the pathways of malate oxidation and phosphorylation in different species of Mycobacteria.

Mycobacterium tuberculosis H37Rv, the slow-growing human pathogenic strain of tubercle bacilli and Mycobacterium smegmatis and Mycobacterium phlei, the fast-growing saprophytes, have shown variations regarding the type of dehydrogenase that initiates malate oxidation in the respiratory chain. M. tuberculosis H37Rv is characterized by having a malate oxidase system (designated MALNAD pathway) in which malate oxidation is mediated by the NAD+-dependent malate dehydrogenase (EC 1.1.1.37) but not by FAD-dependent malate-vitamin K reductase. M. smegmatis possesses a different malate oxidase system (designated MALFAD pathway) in which malate oxidation is exclusively carried out by the FAD-dependent malate-vitamin K reductase because NAD+-dependent malate dehydrogenase is absent in this organism. M. phlei has a mixed system of malate oxidase (designated MALNAD+FAD pathways) in which both the NAD+-and FAD-dependent dehydrogenases take part. In all the three systems, the rest of the electron transport chain is common.     

Nitrate reductase from Spinacea oleracea. FAD and the inactivation by NAD (P) H.

Treatment by p-hydroxymercuribenzoate of nitrate reductase from spinach leaves causes the disappearance of NADH-diaphorase activity and the appearance of an FAD-requirement for the inactivation by NAD(P)H of FNH₂-nitrate reductase. The diaphorase activity of the treated preparation is not affected by incubation with FAD or the addition of this nucleotide to the assay mixture. Conversely, filtration of the native preparation through a column of Sepharose 6B produces the appearance of an FAD-stimulation of the diaphorase activity, but no effect of FAD on the NADH-inactivation was observed. These differences between the FAD-requirement of NADH-diaphorase activity and NADH-inactivation agree with the postulated independence of the two processes.     

An NAD(P) reductase derived from Chlorobium thiosulfa-tophilum: Purification and some properties

A highly purified preparation of an NAD(P) reductase was obtained from Chlorobium thiosulfatophilum and some of its properties were studied. The enzyme possesses FAD as the prosthetic group, and reduces benzyl viologen, 2,6-dichloro-phenolindophenol and cytochromes c, including cytochrome c-555 (C. thiosulfato-philum), with NADPH or NADH as the electron donor. It reduces NADP⁺ or NAD⁺ photosynthetically with spinach chloroplasts in the presence of added spinach ferredoxin. It reduces the pyridine nucleotides with reduced benzyl viologen. The enzyme also shows a pyridine nucleotide transhydrogenase activity. In these reactions, the type of pyridine nucleotide (NADP or NAD) which functions more efficiently with the enzyme varies with the concentration of the nucleotide used; at concentrations lower than approx. 1.0 mM, NADPH (or NADP⁺) is better electron donor (or acceptor), while NADH (or NAD⁺) is a better electron donor (or acceptor) at concentrations higher than approx. 1.0 mM. Reduction of dyes or cytochromes c catalysed by the enzyme is strongly inhibited by NADP⁺, 2′-AMP and and atebrin.     

Properties and reaction mechanism of mitochondrial menadione reductase]

An isolation procedure of mitochondrial menadione reductase from rat liver using an ethanol-ether extraction for solubilization of the enzyme is described. The enzyme was purified 930-fold. The molecular weight of mitochondrial menadione reductase is 62,000. According to spectroscopic and enzymic analysis the prosthetic group of the enzyme was identified as FAD. Mitochondrial menadione reductase is inhibitied by dicumarol and p-chloromecuribenzoate. The enzyme is characterized by a group substrate specificity towards quinones. A high catalytic activity of menadione reductase towards 4-aniline-5-methoxy-1,2-benzoquinone (AMOBQ), and 4-N-(p-sulfoanilino)-5-methoxy-1,2-benzoquinone (AMOBQS) as acceptors was demonstrated. It was shown that the reduction of these orto-benzoquinones by NAD(P) H follows the "ping-pong" kinetics. The kinetic constants for NAD(P)H,AMOBQ and and AMOBQS were determined.     

Preparation and some properties of homogeneous Neurospora crassa assimilatory NADPH-nitrite reductase.

The Neurospora crassa assimilatory NADPH-nitrite reductase (NAD(P)H: nitrite oxidoreductase, EC 1.6.6.4), which catalyzes the NADPH-dependent formation of ammonia from nitrite, has been purified to homogeneity as judged by polyacrylamide gel electrophoresis. The specific activity of the purified enzyme is 26.9 mumol nitrite reduced/min per mg protein, which corresponds to a turnover number of 7800 min(-1). The enzyme also has associated NADH-nitrite reductase, NADPH-hydroxylamine reductase and NADH-hydroxylamine reductase activities. The stoichiometry of 3 mol NADPH oxidized per mol nitrite reduced and ammonia formed has been confirmed. The visible absorption spectrum of the nitrite reductase reveals maxima at 280,390 (Soret) and 580 (alpha) nm. The latter bands are indicative of the occurrence of siroheme as a prosthetic group. The A280nm/A390nm ratio of 7.0 and the Soret/alpha ratio of 3.8 are compatible with values reported for other purified siroheme-containing enzymes. These results are discussed in terms of the comparative biochemistry of various enzymes involved in nitrite, hydroxylamine and sulfite metabolism in Neurospora crassa and other organisms.     

Purification and crystallization of rat liver NAD(P)H:(quinone-acceptor) oxidoreductase by cibacron blue affinity chromatography: identification of a new and potent inhibitor

Cytosolic NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) is a widely distributed, FAD-containing enzyme that catalyzes the obligatory two-electron reduction of quinones. Cibacron Blue is an inhibitor of this enzyme comparable in potency to dicoumarol. Pure quinone reductase was obtained from the livers of Sudan II (1-[2,4-dimethylphenylazo]-2-naphthol)-treated rats in a single step by Cibacron Blue-agarose chromatography. Cibacron Blue is a competitive inhibitor with respect to NADH (Ki = 170 nM) and is a noncompetitive inhibitor with respect to menadione (Ki = 540 nM). Addition of Cibacron Blue to quinone reductase resulted in a decrease and red shift of the enzyme-bound FAD peak at 450 nm. The titration of the absorbance changes for both FAD and Cibacron Blue could be fitted to curves describing an equilibrium binding equation with a KD of 300 nM and one binding site per enzyme subunit. Furthermore, the Cibacron Blue difference spectrum that resulted from binding to quinone reductase was abolished by dicoumarol. Significant amino acid homology between quinone reductase and the nucleotide binding regions of enzymes that bind to Cibacron Blue was found. These data indicate that Cibacron Blue is a useful ligand for the purification of quinone reductase and a new probe for its NAD(P)H binding site. Conditions for crystallizing rat liver quinone reductase are also described.     

The crystal structure of NAD(P)H oxidase from Lactobacillus sanfranciscensis: insights into the conversion of O2 into two water molecules by the flavoenzyme

The FAD-dependent NAD(P)H oxidase from Lactobacillus sanfrancisensis (L.san-Nox2) catalyzes the oxidation of 2 equivalents of either NADH or NADPH and reduces 1 equivalent of O(2) to yield 2 equivalents of water. During steady-state turnover only 0.5% of the reducing equivalents are detected in solution as hydrogen peroxide, suggesting that it is not released from the enzyme after the oxidation of the first equivalent of NAD(P)H and reaction with O(2). Here we report the crystal structure of L.san-Nox2 to 1.8 A resolution. The enzyme crystallizes as a dimer with each monomer consisting of a FAD binding domain (residues 1-120), a NAD(P)H binding domain (residues 150-250), and a dimerization domain (residues 325-451). The electron density for the redox-active Cys42 residue located adjacent to the si-face FAD is consistent with oxidation to the sulfenic acid (Cys-SOH) state. The side chain of Cys42 is also observed in two conformations; in one the sulfenic acid is hydrogen bonded to His10 and in the other it hydrogen bonds with the FAD O2' atom. Surprisingly, the NAD(P)H binding domains each contain an ADP ligand as established by electron density maps and MALDI-TOF analysis of the ligands released from heat-denatured enzyme. The ADP ligand copurifies with the enzyme, and its presence does not inhibit enzyme activity. Consequently, we hypothesize that either NADPH or NADH substrates bind via a long channel that extends from the enzyme exterior and terminates at the FAD re-face. A homology model of the NADH oxidase from Lactococcus lactis (L.lac-Nox2) was also generated using the crystal structure of L.san-Nox2, which reveals several important similarities and differences between the two enzymes. HPLC analysis of ligands released from denatured L.lac-Nox2 indicates that it does not bind ADP, which correlates with the specificity of the enzyme for oxidation of NADH.     

Paradoxical effects of copper and manganese on brain mitochondrial function

Defects in the mitochondrial genome have been associated with Parkinson's and Alzheimer's disease, and apoptosis can be triggered by the presence of energetically compromised mitochondria. Thus, in this study we have examined whether the divalent cations Cu2+ and Mn2+ could influence mitochondrial function in vitro. Mitochondrial electron transport was dose and time dependently reduced by Cu2+ to a greater extent with succinate as a substrate. Following a 60 min preincubation period, Mn2+ dose dependently inhibited electron transport to a greater extent with lactate and malate. In contrast, paradoxical effects were seen following a 5 min preincubation period with Mn2+. Cu2+ dose-dependently reduced NADH-dependent lactate dehydrogenase (LDH) activity, with almost complete inhibition apparent at 10 microM. An initial induction of LDH by 10 microM Mn2+ was partially reversed by higher concentrations of the metal. Cu2+ dose-dependently reduced flavin adenine dinucleotide (FAD)-dependent monoamine oxidase A (MAO-A) activity in a time-independent manner, with an IC50 value approximately 20 microM, whereas Mn2+ had no effect. In conclusion, it is proposed that Cu2+ and Mn2+ have differential effects on nicotinamide adenine dinucleotide (NAD) and FAD-dependent mitochondrial enzymes at the level of the essential cofactors. Cu2+ appears to exert an inhibitory effect on both NAD and FAD-dependent enzymes, but predominantly against the latter, including MAO-A and succinate dehydrogenase. The complex responses to Mn2+ may be due to dose-related effects on the interconversion of NAD and NADH and reversible enzymatic reactions employing this nucleotide cofactor.     

Lipid composition and (Na+ + K+)-ATPase activity in rat lens during triparanol-induced cataract formation

In vitro complementation of the soluble assimilatory NAD(P)H-nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) was attained by mixing cell-free preparations of Chlamydomonas reinhardii mutant 104, uniquely possessing nitrate-inducible NAD(P)H-cytochrome c reductase, and mutant 305 which possesses solely the nitrate-inducible FMNH2- and reduced benzyl viologen-nitrate reductase activities. Full activity and integrity of NAD(P)H-cytochrome c reductase from mutant 104 and reduced benzyl viologen-nitrate reductase from mutant 305 are needed for the complementation to take place. A constitutive and heat-labile molybdenum-containing cofactor, that reconstitutes the NAD(P)H-nitrate reductase activity of nit-1 Neurospora crassa but is incapable of complementing with 104 from C. reinhardii, is present in the wild type and 305 algal strains. The complemented NAD(P)H-nitrate reductase has been purified 100-fold and was found to be similar to the wild enzyme in sucrose density sedimentation, molecular size, pH optimum, kinetic parameters, substrate affinity and sensitivity to inhibitors and temperature. From previous data and data presented in this article on 104 and 305 mutant activities, it is concluded that C. reinhardii NAD(P)H-nitrate reductase is a heteromultimeric complex consisting of, at least, two types of subunits separately responsible for the NAD(P)H-cytochrome c reductase and the reduced benzyl viologen-nitrate reductase activities.