2-Aminobenzoyl-CoA monooxygenase/reductase, a novel type of flavoenzyme. Studies on the stoichiometry and the course of the reaction

The reaction catalyzed by 2-aminobenzoyl-coenzyme-A monooxygenase/reductase from a denitrifying Pseudomonas sp. has been investigated. 2-Aminobenzoyl-CoA and 2-amino[carboxy-14C]benzoyl-CoA were synthesized enzymatically using 2-aminobenzoyl-CoA synthetase from the same organism. The product was purified by chromatography and characterized by ultraviolet/visible and 1H-NMR spectroscopy. The conversion of 2-aminobenzoyl-CoA catalyzed by the monooxygenase/reductase requires NADH and oxygen, and yields at least two different products depending on the relative concentration of NADH. At [NADH] less than Km (40 microM), i.e. [NADH]/[2-aminobenzoyl-CoA] approximately 0.02-0.05, the main product is probably a hydroxylated derivative of 2-aminobenzoyl-CoA, which is characterized by an absorbance maximum around 375 nm. When [NADH]/[2-aminobenzoyl-CoA] approximately 2-5, the predominant product is a non-aromatic coenzyme A thioester (lambda max approximately 320 nm). The stoichiometry in this case is 2.1-2.4 mol NADH oxidized (mol oxygen consumed)-1 (mol 2-aminobenzoyl-CoA metabolized)-1. The product is extremely unstable in the acidic pH range and undergoes decarboxylation in a few minutes at pH less than 5. Some degree of stabilisation is obtained upon reduction with sodium borohydride, probably resulting in a further reduced non-aromatic coenzyme-A thioester.     

A continuous spectrophotometric assay method for peptidylarginine deiminase type 4 activity

A simple, continuous spectrophotometric assay for peptidylarginine deiminase (PAD) is described. Deimination of peptidylarginine results in the formation of peptidylcitrulline and ammonia. The ammonia released during peptidylarginine hydrolysis is coupled to the glutamate-dehydrogenase-catalyzed reductive amination of alpha-ketoglutarate to glutamate and reduced nicotinamide adenine dinucleotide (NADH) oxidation. The disappearance of absorbance at 340nm due to NADH oxidation is continuously measured. The specific activity obtained by this new protocol for highly purified human PAD is comparable to that obtained by a commonly used colorimetric procedure, which measures the ureido group of peptidylcitrulline by coupling with diacetyl monoxime. The present continuous spectrophotometric method is highly sensitive and accurate and is thus suitable for enzyme kinetic analysis of PAD. The Ca(2+) concentration for half-maximal activity of PAD obtained by this method is comparable to that previously obtained by the colorimetric procedure.     

Distribution, purification, and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes.

Phenylalanine dehydrogenase (L-phenylalanine:NAD oxidoreductase, deaminating; EC 1.4.1.-) was found in various thermophilic actinomycetes. We purified the enzyme to homogeneity from Thermoactinomyces intermedius IFO 14230 by heat treatment and by Red Sepharose 4B, DEAE-Toyopearl, Sepharose CL-4B, and Sephadex G-100 chromatographies with a 13% yield. The relative molecular weight of the native enzyme was estimated to be about 270,000 by gel filtration. The enzyme consists of six subunits identical in molecular weight (41,000) and is highly thermostable: it is not inactivated by incubation at pH 7.2 and 70 degrees C for at least 60 min or in the range of pH 5 to 10.8 at 50 degrees C for 10 min. The enzyme preferably acts on L-phenylalanine and its 2-oxo analog, phenylpyruvate, in the presence of NAD and NADH, respectively. Initial velocity and product inhibition studies showed that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism. The Km values for L-phenylalanine, NAD, phenylpyruvate, NADH, and ammonia were 0.22, 0.078, 0.045, 0.025, and 106 mM, respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH was exclusively transferred to the substrate.     

Purification and Kinetics of Higher Plant NADH:Nitrate Reductase

Squash cotyledon (Cucurbita pepo L.) NADH:nitrate reductase (NR) was purified 150-fold with 50% recovery by a single step procedure based on the affinity of the NR for blue-Sepharose. Blue-Sepharose, which is prepared by direct coupling of Cibacron blue to Sepharose, appears to bind squash NR at the NADH site. The NR can be purified in 2 to 3 hours to a specific activity of 2 μmol of NADH oxidized/minute • milligram of protein. Corn (Zea mays L.) leaf NR was also purified to a specific activity of 6.9 μmol of NADH oxidized/minute • milligram of protein using a blue-Sepharose affinity step. The blue-Sepharose method offers the advantages of a rapid purification of plant NR to a high specific activity with reasonable recovery of total activity.

The kinetic mechanism of higher plant NR was investigated using these highly purified squash and corn NR preparations. Based on initial velocity and product inhibition studies utilizing both enzymes, a two-site ping-pong mechanism is proposed for NR. This kinetic mechanism incorporates the concept of the reduced NR transferring electrons from the NADH site to a physically separated nitrate site.

     

Acetyl-CoA-dependent elongation of fatty acids in Mycobacterium smegmatis.

An enzyme system of Mycobacterium smegmatis catalyzing the elongation of medium-chain fatty acids with acetyl-CoA was obtained free from de novo fatty acid synthetase by ammonium sulfate fractionation. The system was resolved by gel filtration and DEAE-cellulose chromatography into three fractions, all of which were required for reconstitution of the elongation activity. The three fractions were highly purified enoyl-CoA hydratase, highly purified 3-hydroxyacyl-CoA dehydrogenase, and a fraction containing both enoyl-CoA reductase and thiolase. The reconstituted system was avidin-insenstive, required NADH as a sole hydrogen donor, and was sensitive to pCMB, but not to N-ethylmaleimide or monoiodoacetate. Decanoyl-CoA and octanoyl-CoA were the best primers for the elongation system. When decanoyl-CoA was used as the primer, the major product was found to be a lauroyl derivative (probably lauroyl-CoA). Evidence was obtained suggesting that acyl-CoA dehydrogenase, catalyzing the first step of beta-oxidation, was not functional in the elongation system.     

A continuous 96-well plate spectrophotometric assay for branched-chain amino acid aminotransferases

A new, continuous 96-well plate spectrophotometric assay for the branched-chain amino acid aminotransferases is described. Transamination of L-leucine with alpha-ketoglutarate results in formation of alpha-ketoisocaproate, which is reductively aminated back to L-leucine by leucine dehydrogenase in the presence of ammonia and NADH. The disappearance of absorbance at 340 nm due to NADH oxidation is measured continuously. The specific activities obtained by this procedure for the highly purified human mitochondrial and cytosolic isoforms of BCAT compare favorably with those obtained by a commonly used radiochemical procedure, which measures transamination between alpha-ketoiso[1-14C]valerate and L-isoleucine. Due to the presence of glutamate dehydrogenase substrates (alpha-ketoglutarate, ammonia, and NADH) and L-leucine (an activator of glutamate dehydrogenase) in the standard assay mixture, interference with the measurement of BCAT activity in tissue homogenates by glutamate dehydrogenase is observed. However, by limiting the amount of ammonia and including the inhibitor GTP in the assay mixture, the interference from the glutamate dehydrogenase reaction is minimized. By comparing the rate of loss of absorbance at 340 nm in the modified spectrophotometric assay mixture containing leucine dehydrogenase to that obtained in the modified spectrophotometric assay mixture lacking leucine dehydrogenase, it is possible to measure BCAT activity in microliter amounts of rat tissue homogenates. The specific activities of BCAT in homogenates of selected rat tissues obtained by this method are comparable to those obtained previously by the radiochemical procedure.     

The purification and characterization of acetoacetyl-coenzyme A reductase from Azotobacter beijerinckii

A soluble acetoacetyl-CoA reductase (EC 1.1.1.36) was purified 54-fold from Azotobacter beijerinckii N.C.I.B. 9067 and the reaction product identified as d(-)-beta-hydroxybutyryl-CoA. The Michaelis constants for acetoacetyl-CoA, NADPH and NADH were determined and the reaction rate was found to be some fivefold greater with NADPH than with NADH. At neutral pH the equilibrium greatly favours the formation of the reduced product. Substrate specificity was in the order: acetoacetyl-CoA>acetoacetylpantetheine>acetoacetyl-(acyl-carrier protein). The enzyme possesses a functional thiol group, suffers inactivation by oxygen and is inhibited by thiol-blocking reagents. Inhibition by p-chloromercuribenzoate is reversed by excess of dithiothreitol, which also protects the enzyme from inactivation by oxygen.     

Optimized expression and purification of toluene 4-monooxygenase hydroxylase

Toluene 4-monooxygenase is a four-protein complex that catalyzes the O(2)- and NADH-dependent oxidation of toluene to p-cresol. The influence of various expression systems on the host cell growth characteristics, purified protein yields, and specific activity of the hydroxylase (T4moH) component of the complex was evaluated by considering the cell mass obtained per liter of fermentation culture medium, the purified protein obtained per gram of cell mass, and the specific activity of purified T4moH. The specific activity of purified T4moH was determined to be 1200-1250 nmol of p-cresol formed per minute per milligram of T4moH in air-saturated 50 mM phosphate buffer, pH 7.5, at 25 degrees C in the presence of optimal concentrations of the other protein components of the complex, saturating toluene (5.8 mM at 25 degrees C), and saturating NADH (1 mM). This value was obtained for T4moH purified from several different expression systems and apparently represents the maximal specific activity of the enzyme complex for toluene hydroxylation. By manipulation of vectors and gene inserts to eliminate adventitious catalytic turnover of NADH, up to 60-fold increase in the volumetric yield of T4moH activity was obtained from recombinant fermentations in Escherichia coli BL21(DE3).     

Genetic identification and purification of the respiratory NADH dehydrogenase of Escherichia coli

Escherichia coli membrane particles were solubilized with potassium cholate. An NADH:ubiquinone oxidoreductase was resolved by hydroxylapatite chromatography of the solubilized material. This enzyme has been identified as the respiratory NADH dehydrogenase since it is absent in chromatograms of solubilized material from an ndh mutant strain. Such mutants lack membrane-bound NADH oxidase activity and have previously been shown to have an inactive NADH dehydrogenase complex [Young, I. G., & Wallace, B. J. (1976) Biochim. Biophys. Acta 449, 376-385]. The respiratory NADH dehydrogenase was amplified 50- to 100-fold in vivo by using multicopy plasmid vectors carrying the ndh gene and then purified to homogeneity on hydroxylapatite. Hydroxylapatite chromatography of cholate-solubilized material from genetically amplified strains purified the enzyme approximately 800- to 100-fold relatively to the activity in wild-type membranes. By use of a large-scale purification procedure, 50-100 mg of protein with a specific activity of 500-600 mumol of reduced nicotinamide adenine dinucleotide oxidized min-1 mg-1 at pH 7.5, 30 degrees C, was obtained. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme showed that the enzyme consists of a single polypeptide with an apparent Mr of 45 000.     

Purification and properties of alpha-ketoadipate reductase, a newly discovered enzyme from human placenta.

A newly discovered enzyme, α-ketoadipate reductase, has been purified 1000-fold from human placenta. This enzyme catalyzes the following reaction: α-ketoadipate + NADH + H⁺ → α-hydroxyadipate + NAD. The enzyme has an estimated molecular weight of 95,000 on gel filtration and an isoelectric point at pH 7.0 on electrofocusing. Several forms of the enzyme were isolated during purification. The pH optimum for the major form was 6.3. The reaction product of α-ketoadipate reductase was identified as α-hydroxyadipate by comparison of the enzyme product with chemically prepared α-hydroxyadipate. Studies of the reaction stoichiometry indicated that equimolar quantities of NADH and α-ketoadipate were used in the synthesis of an equivalent quantity of α-hydroxyadipate. Under conditions where the remaining lactate dehydrogenase and malate dehydrogenase were completely inhibited without affecting the α-ketoadipate reductase activity, it was found that α-ketoadipate reductase was highly specific for α-ketoadipate as substrate. NADPH could not substitute for NADH. Initial velocity experiments showed that NADH was an uncompetitive substrate inhibitor.     

Isolation,characterization and electron microscopic single particle analysis of the NADH:ubiquinone oxidoreductase (complex I) from the hyperthermophilic eubacterium Aquifex aeolicus

The proton-translocating NADH:ubiquinone oxidoreductase (complex I) has been purified from Aquifex aeolicus, a hyperthermophilic eubacterium of known genome sequence. The purified detergent solubilized enzyme is highly active above 50 degrees C. The specific activity for electron transfer from NADH to decylubiquinone is 29 U/mg at 80 degrees C. The A. aeolicus complex I is completely sensitive to rotenone and 2-n-decyl-quinazoline-4-yl-amine. SDS polyacrylamide gel electrophoresis shows that it may contain up to 14 subunits. N-terminal amino acid sequencing of the bands indicates the presence of a stable subcomplex, which is composed of subunits E, F, and G. The isolated complex is highly stable and active in a temperature range from 50 to 90 degrees C, with a half-life of about 10 h at 80 degrees C. The activity shows a linear Arrhenius plot at 50-85 degrees C with an activation energy at 31.92 J/mol K. Single particle electron microscopy shows that the A. aeolicus complex I has the typical L-shape. However, visual inspection of averaged images reveals many more details in the external arm of the complex than has been observed for complex I from other sources. In addition, the angle (90 degrees ) between the cytoplasmic peripheral arm and the membrane intrinsic arm of the complex appears to be invariant.     

Non-invasive methods for determination of cellular growth inPodophyllum hexandrum suspension cultures

Culture conductivity and on-line NADH fluorescence were used to measure cellular growth in plant cell suspension cultures ofPodophyllum hexandrum. An inverse correlation between dry cell weight and medium conductivity was observed during shake flask cultivation. A linear relationship between dry cell weight and culture NADH fluorescence was obtained during the exponential phase of batch cultivation in a bioreactor under the pH stat (pH 6) conditions. It was observed that conductivity measurement were suitable for biomass characterisation under highly dynamic uncontrolled shake flask cultivation conditions. However, if the acid/alkali feeding is done for pH control the conductivity measurement could not be applied. On the other hand the NADH fluorescence measurement allowed online-in situ biomass monitoring of rather heterogenous plant cell suspension cultures in bioreactor even under the most desirable pH stat conditions.     

Studies of the human testis. XX--Effect of NADH on 17 alpha-hydroxypregnene C17-20 lyase activity

A study of the cofactor requirements of C17-20 lyase was carried out using human testis tissue obtained at the time of orchiectomy for treatment of prostatic carcinoma. Washed microsomal fractions were prepared from frozen human testes using a KCl containing buffer. The preparation revealed dose-dependent activity of C17-20 lyase in the presence of either NADPH or commercial or purified NADH. The Km of NADH for the enzyme was of the order of 10(-3) M and the Km of NADPH was determined as 1.6 X 10(-5) M. NADH also provided synergistic enhancement of NADPH-mediated lyase activity, and decreased the Km of NADPH for the lyase but did not change the Vmax of NADPH-mediated lyase activity. Carbon monoxide inhibited both NADH and NADPH-mediated lyase activities indicating that both activities are catalyzed by cytochrome P-450. Cations including Ca2+, Mg2+ and Mn2+ were found to inhibit the NADPH-mediated lyase activity but enhanced the lyase activity in the presence of NADH. The results indicate both the presence of NADH-mediated C17-20 lyase activity and the synergistic effect of NADH on NADPH-mediated lyase activity in the human testis.     

Isolation and characterization of a tumor-associated NADH oxidase (tNOX) from the HeLa cell surface.

Cell-surface-located, drug-responsive and tumor-associated NADH oxidase (tNOX) proteins were purified and characterized from HeLa cells. The proteins isolated exhibited NADH oxidase activity inhibited by capsaicin and were resistant to heating and to protease digestion. The activity was purified 200- to 500-fold to provide apparently homogeneous gel bands for N-terminal sequencing using three different protocols. All three protocols involved heat (50 degrees C) and proteinase K treatment. Recovery of the total NADH oxidase activity was 86% and inhibition by capsaicin was 60 to 80%. After 450-fold purification, a 52-kDa component was obtained as a single gel band that retained the capsaicin-inhibited NADH oxidase activity. Amino acid composition and partial amino acid sequences were obtained. The partial amino acid sequences were used to generate peptide antisera. Both the peptide antisera and polyclonal antisera to the 52-kDa component immunoprecipitated capsaicin-inhibited NADH oxidase activity and reacted with 52-, 34-, and 17-kDa components on Western blots from different steps of the purification. The tNOX protein exhibited immunological cross-reactivity and amino acid sequence identity with tNOX cloned from a HeLa cDNA library using a monoclonal antibody to tNOX from sera of cancer patients. The results provide a direct sequence link between tNOX of the HeLa cell surface and the cloned tNOX representative of patient sera. The tNOX form from the surface of HeLa cells yielded N-terminal sequence consistent with a coidentity of the cell surface and serum forms of the two activities.     

Polysaccharide production benefits dry storage survival of the biocontrol agent Pseudomonas fluorescens S11:P:12 effective against several maladies of stored potatoes

Pseudomonas fluorescens S11:P:12 (NRRL B-21133) is a biological control agent able to suppress several potato diseases and sprouting. Notably, it produces a polysaccharide during liquid cultivation, and the objective of this work was to determine the role of this material in the bio-control process. First, the polysaccharide was isolated, purified and identified as marginalan, which accumulated to ~3.3 g/L in cultures. The bioactivity of isolated marginalan applied alone or in combination with washed cells of strain S11:P:12 was tested in potato bioassays of dry rot and pink rot suppressiveness and sprout inhibition. Since the formulation and storage of a dried biocontrol product is preferred for commercial use, the impact of marginalan on cell survival during drying and storage was also studied. Washed bacteria formulated with 0–6.6 g/L polysaccharide were either applied to Hyflo granules, then slowly dried for 24 h with airflow at 50–60% relative humidity, or in 1-µL droplets placed in replicate wells of a micro-plate, then quickly dried for 1 h in a biohazard hood. Both Hyflo and micro-plate dry storage results indicated that marginalan significantly reduced cell death after drying, such that the final stable viable cell density was 2.5–5 orders of magnitude greater, respectively, than if no marginalan were included with cells. Marginalan had no significant impact on disease or sprout suppression by strain S11:P:12, and its main benefit to biocontrol was viable cell preservation during drying and storage. When marginalan was formulated with other selected P. fluorescens strains, its benefits to drying and storage survival were again evident (especially after 4°C instead of 25°C storage), but its effects were more subtle than for strain S11:P:12, and dry rot suppression was not impacted.     

Naphthalene dioxygenase: purification and properties of a terminal oxygenase component

Naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816 is a multicomponent enzyme system that oxidized naphthalene to cis-(1R, 2S)-dihydroxy-1,2-dihydronaphthalene. The terminal oxygenase component B was purified to homogeneity by a three-step procedure that utilized ion-exchange and hydrophobic interaction chromatography. The purified enzyme oxidized naphthalene only in the presence of NADH, oxygen, and partially purified preparations of components A and C. An estimated Mr of 158,000 was obtained by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of two subunits with molecular weights of ca. 55,000 and 20,000, indicative of an alpha 2 beta 2 quaternary structure. Absorption spectra of the oxidized enzyme showed maxima at 566 (shoulder), 462, and 344 nm, which were replaced by absorption maxima at 520 and 380 nm when the enzyme was reduced anaerobically by stoichiometric quantities of NADH in the presence of the other two components of the naphthalene dioxygenase system. Component B bound naphthalene. Enzyme-bound naphthalene was oxidized to product upon the addition of components A and C, NADH, and O2. These results, together with the detection of the presence of 6.0 g-atoms of iron and 4.0 g-atoms of acid-labile sulfur per mol of the purified enzyme, suggest that component B of the naphthalene dioxygenase system is an iron-sulfur protein which functions in the terminal step of naphthalene oxidation.     

Fertilizing value of broiler litter: effects of drying and pelletizing

The effects of drying and pelletizing on the properties of broiler chicken litter, obtained from a farm in northwest Spain, were investigated. The drying and pelletizing process reduced among-batch variability in dry matter content, electrical conductivity, urea N, and K, S, Na, Fe, Cu and Cd contents, but increased among-batch variability in total N, ammonium N, nitrate N, total P and pH. N form contents in the pelletized product could be estimated with reasonable accuracy on the basis of dry matter content. Cr, Cu and Cd contents were all significantly lower in the dried pelletized product than in fresh litter, whereas Pb content was significantly higher. The dried pelletized product is of course clearly preferable to the fresh product as regards storage and handling, however, our results suggest a need to optimize the production process with the aim of reducing possible contamination during manufacture, and of minimizing variability in N form contents, P content and pH.     

Purification and characterization of a rotenone-insensitive NADH:Q6 oxidoreductase from mitochondria of Saccharomyces cerevisiae

A mitochondrial NADH:Q6 oxidoreductase has been isolated from cells of Saccharomyces cerevisiae by a simple method involving extraction of the enzyme from the mitochondrial membrane with Triton X-100, followed by chromatography on DEAE-cellulose and blue Sepharose CL-6B. By this procedure a 2000-fold purification is achieved with respect to whole cells or a 150-fold purification with respect to the mitochondrion. The purified NADH dehydrogenase consists of a single subunit with molecular mass of 53 kDa as indicated by SDS/polyacrylamide gel electrophoresis. The enzyme contains FAD, non-covalently linked, as the sole prosthetic group with Em,7.6 = -370 mV and no iron-sulphur clusters. The enzyme is specific for NADH with apparent Km = 31 microM and was found to be inhibited by flavone (I50 = 95 microM), but not by rotenone or piericidin. The purified enzyme can use ubiquinone-2, -6 or -10, menaquinone, dichloroindophenol or ferricyanide as electron acceptors, but at different rates. The greatest turnover of NADH was obtained with ubiquinone-2 as acceptor (2500 s-1). With the natural ubiquinone-6 this value was 500 s-1. The NADH:Q2 oxidoreductase activity shows a maximum at pH 6.2, the NADH:Q6 oxidoreductase activity is constant between pH 4.5-9.0. The amount of enzyme in the cell is subject to glucose repression; it increases slightly when cells, grown on glucose or lactate, enter the stationary phase. The experiments performed so far suggest that the enzyme purified in this study is the external NADH:Q6 oxidoreductase, bound to the mitochondrial inner membrane and that it is involved in the oxidation of cytosolic NADH. The relation of this enzyme with respect to various other NADH dehydrogenases from yeast and plant mitochondria is discussed.     

Pilot plant scale extraction of alginates from Macrocystis pyrifera 4. Conversion of alginic acid to sodium alginate, drying and milling

The last three steps of the alginate production process were studied:conversion of alginic acid to sodium alginate, drying, and milling. Threemethods were used to follow the conversion reaction: measuring the pH (a) intheethanol-water liquid of the reaction mixture, (b) after dissolving a sample ofthe fiber taken from the reaction mixture, (c) after dissolving the driedsodiumalginate obtained from the reaction. To obtain a neutral dried sodium alginate,in the first method the pH should be adjusted to 9, and in the second the pHshould be adjusted to 8. The best method to control the reaction was todissolvea sample of the fiber and adjust the pH to 8. The best proportion to reach thecritical point, where pH just begins to rise, was 0.25 parts of sodiumcarbonateto 1 part of alginate in the initial dry algae. A pH above 7 may produce abreakdown of the molecule, reducing significantly the viscosity of the finalalginate. Four different temperatures were used to dry the alginate: 50, 60,70,and 80 °C. Drying time to reach 12% moisture ranged from 1.5h at 80 °C to 3 h at 50°C. The best drying temperature was 60 °C for2.5 h. The effect of drying temperature on alginate viscosity wasdependent on the alginate type. Low and medium viscosity alginates were notsignificantly affected, but alginate with high viscosity was reduced by 40 to54% using the temperature range of 60 to 80 °C. A fixed hammermill was used to reduce the particle size of the dried sodium alginate.Particlesize measurements showed that after a first milling the product contained 76%large particles (20–60 mesh) and 24% fine particles (80–120 mesh).After a third milling the product still contained 42.9% large particles. Nosignificant effect was found on alginate viscosity because of the millingsteps.     

Purification and properties of NADH dehydrogenase from a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius

An NADH dehydrogenase was purified to electrophoretical homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium optimally growing at pH 2-3 and 75 degrees C. A 2,100-fold purification was achieved. The purified enzyme is an acidic protein with an isoelectric point of 5.6 and a molecular weight of 95,000, consisting of two 50,000-dalton subunits. The enzyme showed an absorption spectrum characteristic of flavoproteins, with maxima at 272, 372, and 448 nm. The enzyme is highly thermostable, is specific for NADH as an electron donor, and is capable of using 2,6-dichlorophenolindophenol, ferricyanide, benzoquinone, and naphthoquinone as electron acceptors. Though at a low rate, caldariellaquinone, a unique and sole benzothiophenequinone in the genus Sulfolobus, was also reduced by the enzyme, suggesting that the enzyme is a possible member of the respiratory chain of the thermoacidophilic archaebacterium.