Pyrogallol-to-phloroglucinol conversion and other hydroxyl-transfer reactions catalyzed by cell extracts of Pelobacter acidigallici.

Permeabilized cells and cell extracts of Pelobacter acidigallici catalyzed the conversion of pyrogallol (1,2,3-trihydroxybenzene) to phloroglucinol (1,3,5-trihydroxybenzene) in the presence of 1,2,3,5-tetrahydroxybenzene. Pyrogallol consumption by resting cells stopped after lysis by French press or mild detergent (cetyltrimethylammonium bromide [CTAB]) treatment. Addition of 1,2,3,5-tetrahydroxybenzene to the assay mixture restored pyrogallol consumption and led to stoichiometric phloroglucinol accumulation. The stoichiometry of pyrogallol conversion to phloroglucinol was independent of the amount of tetrahydroxybenzene added. The tetrahydroxybenzene concentration limited the velocity of the transhydroxylation reaction, which reached a maximum at 1.5 mM tetrahydroxybenzene (1 U/mg of protein). Transhydroxylation was shown to be reversible. The equilibrium constant of the reaction was determined, and the free-energy change (delta G degree') of phloroglucinol formation from pyrogallol was calculated to be -15.5 kJ/mol. Permeabilized cells and cell extracts also catalyzed the transfer of hydroxyl moieties between other hydroxylated benzenes. Tetrahydroxybenzene and hydroxyhydroquinone participated as hydroxyl donors and as hydroxyl acceptors in the reaction, whereas pyrogallol, resorcinol, and phloroglucinol were hydroxylated by both donors. A novel mechanism deduced from these data involves intermolecular transfer of the hydroxyl moiety from the cosubstrate (1,2,3,5-tetrahydroxybenzene) to the substrate (pyrogallol), thus forming the product (phloroglucinol) and regenerating the cosubstrate.     

Transhydroxylase of Pelobacter acidigallici: a molybdoenzyme catalyzing the conversion of pyrogallol to phloroglucinol

Trihydroxybenzenes are degraded anaerobically through the phloroglucinol pathway. In Pelobacter acidigallici as well as in Pelobacter massiliensis, pyrogallol is converted to phloroglucinol in the presence of 1,2,3,5-tetrahydroxybenzene by intermolecular hydroxyl transfer. The enzyme catalyzing this reaction was purified to chromatographic and electrophoretic homogeneity. Gel filtration and electrophoresis revealed a heterodimer structure with an apparent molecular mass of 127 kDa for the native enzyme and 86 kDa and 38 kDa, respectively, for the subunits. The enzyme was not sensitive to oxygen. HgCl₂, p-chloromercuribenzoic acid, and CuCl₂ inhibited strongly the reaction indicating an essential function of SH-groups. Transhydroxylase had a pH-optimum of 7.0 and a pI of 4.1. The apparent temperature optimum was in the range of 53°C to 58°C. The activation energy for the conversion of pyrogallol and 1,2,3,5-tetrahydroxybenzene to phloroglucinol and tetrahydroxybenzene was 31.4 kJ per mol. Purified enzyme exhibited a specific activity of 3.1 mol. m⁻¹ mg⁻¹ protein and an apparent K_m for pyrogallol and 1,2,3,5-tetrahydroxybenzene of 0.70 mM and 0.71 mM, respectively. The enzyme was found to contain per mol heterodimer 1.1 mol molybdenum, 12.1 mol iron and 14.5 mol acid-labile sulfur. Requirement for molybdenum for transhydroxylating enzyme activity was proven also by cultivation experiments. No hints for the presence of flavins were obtained. The results presented here support the hypothesis that a redox reaction is involved in this intermolecular hydroxyl transfer.     

The molybdenum site in the xanthine oxidase-related aldehyde oxidoreductase from Desulfovibrio gigas and a catalytic mechanism for this class of enzymes

 The crystal structure analysis of the aldehyde oxidoreductase from Desulfovibrio gigas was exceptionally revealing with regard to the ligands and structure of the molybdenum site and the mechanism of the hydroxylation reaction catalyzed. The metal is pentacoordinated by two sulfurs of the cis–dithiolene group of the molybdopterin cofactor and by facially arranged sulfido, oxo and water ligands. The latter is in hydrogen-bonding contact with the carboxylate group of Glu 869 and the hydroxyl group of an isopropanol molecule, a substrate analogue inhibitor. This steric arrangement strongly suggests a mechanism for the reductive half-cycle of the reaction with Glu 869 as the base, the metal-bound water as the source of the transferred hydroxyl group, and the sulfido group as the hydride acceptor. The geometry and the proposed mechanism are in agreement with density functional calculations on a model of the molybdenum site. In the oxidative half-reaction, electrons are withdrawn from Mo^rv through the rigidly held pterin ring system, via the iron-sulfur clusters, to the protein surface. Received: 25 June 1997 / Accepted: 20 August 1997     

Novel bacterial molybdenum and tungsten enzymes: three-dimensional structure, spectroscopy, and reaction mechanism

The molybdenum enzymes 4-hydroxybenzoyl-CoA reductase and pyrogallol-phloroglucinol transhydroxylase and the tungsten enzyme acetylene hydratase catalyze reductive dehydroxylation reactions, i.e., transhydroxylation between phenolic residues and the addition of water to a triple bond. Such activities are unusual for this class of enzymes, which carry either a mononuclear Mo or W center. Crystallization and subsequent structural analysis by high-resolution X-ray crystallography has helped to resolve the reaction centers of these enzymes to a degree that allows us to understand the interaction of the enzyme and the respective substrate(s) in detail, and to develop a concept for the respective reaction mechanism, at least in two cases.     

Sequential Transhydroxylations Converting Hydroxyhydroquinone to Phloroglucinol in the Strictly Anaerobic, Fermentative Bacterium Pelobacter massiliensis

The recently isolated fermenting bacterium Pelobacter massiliensis is the only strict anaerobe known to grow on hydroxyhydroquinone (1,2,4-trihydroxybenzene) as the sole source of carbon and energy, converting it to stoichiometric amounts of acetate. In this paper, we report on the enzymatic reactions involved in the conversion of hydroxyhydroquinone and pyrogallol (1,2,3-trihydroxybenzene) to phloroglucinol (1,3,5-trihydroxybenzene). Cell extracts of P. massiliensis transhydroxylate pyrogallol to phloroglucinol after addition of 1,2,3,5-tetrahydroxybenzene (1,2,3,5-TTHB) as cosubstrate in a reaction identical to that found earlier with Pelobacter acidigallici (A. Brune and B. Schink, J. Bacteriol. 172:1070-1076, 1990). Hydroxyhydroquinone conversion to phloroglucinol is initiated in cell extracts without an external addition of cosubstrates. It involves a minimum of three consecutive transhydroxylation reactions characterized by the transient accumulation of two different TTHB isomers. Chemical synthesis of the TTHB intermediates allowed the resolution of the distinct transhydroxylation steps in this sequence. In an initial transhydroxylation, the hydroxyl group in the 1-position of a molecule of hydroxyhydroquinone is transferred to the 5-position of another molecule of hydroxyhydroquinone to give 1,2,4,5-TTHB and resorcinol (1,3-dihydroxybenzene) as products. Following this disproportionation of hydroxyhydroquinone, the 1,2,4,5-isomer is converted to 1,2,3,5-TTHB, an enzymatic activity present only in hydroxyhydroquinone-grown cells. Finally, phloroglucinol is formed from 1,2,3,5-TTHB by transfer of the 2-hydroxyl group to either hydroxyhydroquinone or resorcinol. The resulting coproducts are again cosubstrates in earlier reactions of this sequence. From the spectrum of hydroxybenzenes transhydroxylated by the cell extracts, the minimum structural prerequisites that render a hydroxybenzene a hydroxyl donor or acceptor are deduced.     

Characterization of the pyrogallol-phloroglucinol isomerase of Eubacterium oxidoreducens.

Cell extracts of Eubacterium oxidoreducens, in the presence of dimethyl sulfoxide, catalyzed the conversion of pyrogallol to phloroglucinol with methyl sulfide as a product. The isomerization reaction also proceeded when 1,2,3,5-benzenetetrol was present rather than dimethyl sulfoxide. An assay to quantitate this activity was developed. The assay followed the disappearance of 1,2,4-benzenetriol as determined colorimetrically after incubation with sodium molybdate at neutral pH. The products of this reaction were resorcinol and 2,6-dihydroxyquinone. The enzyme(s) catalyzing this reaction was purified fivefold from cells grown on gallate plus H2. The purification procedure involved treatment with 40% acetone, precipitation with ammonium sulfate, DEAE-cellulose chromatography, concentration by ultrafiltration (molecular weight cutoff, greater than 100,000), and hydroxylapatite chromatography. This preparation had a specific activity of 14.7 mumol/min per mg of protein and a pH optimum of about 7.3. It was strongly inhibited by p-chloromercuribenzoate. The mechanism of the reaction involved oxidation of the pyrogallol followed by introduction of water. The benzenetetrol intermediate was then reduced and dehydrated to phloroglucinol.     

The inactivation of phenylalanine hydroxylase by 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine and the aerobic oxidation of the latter. The effects of catalase, dithiothreitol and reduced nicotinamide–adenine dinucleotide

1. Phenylalanine hydroxylase is inhibited by its cofactor, 6,7-dimethyltetrahydropterin. The rate of inactivation, which is irreversible, increases with the concentration of cofactor. 2. Catalase, in sufficient amount relative to cofactor, prevents this inactivation. More tyrosine is formed in the presence of added catalase. 3. Dithiothreitol in the presence of liver extract also prevents inactivation of the enzyme by the cofactor and stimulates hydroxylation of phenylalanine, probably by protecting the cofactor from oxidation and regenerating it from a dihydropterin reaction product. Dithiothreitol restores linearity of rate at very low enzyme concentrations. 4. Dimethyltetrahydropterin is unstable when the solution is exposed to air but is stabilized by dithiothreitol the aerobic oxidation of which is greatly accelerated by dimethyltetrahydropterin. 5. NADH together with liver extract stabilizes the cofactor but not phenylalanine hydroxylase. 6. It is suggested that either hydrogen peroxide or an organic peroxide formed by oxidation in air of the cofactor is the substance attacking phenylalanine hydroxylase, dithiothreitol and cofactor.     

A radiometric assay for kynurenine 3-hydroxylase based on the release of 3H2O during hydroxylation of L-[3,5-3H]kynurenine.

A rapid and sensitive assay for kynurenine 3-hydroxylase (KH) has been developed. This radiometric assay is based on the enzymatic synthesis of tritiated water from L-[3,5-3H]kynurenine during the hydroxylation reaction. Radiolabeled water is quantified following selective adsorption of the isotopic substrate and its metabolite with activated charcoal. The assay is suitable for detecting 0.1 pmol enzyme activity per minute per milligram protein in tissues displaying low levels of the enzyme. The amount of water produced in the reaction, as calculated from the tritium released, was stoichiometric with the 3-hydroxykynurenine product detected by HPLC. Rat liver KH was characterized by cofactor specificity and kinetic parameters. NADPH was preferred over NADH as coreductant in the reaction. Tetrahydrobiopterin was not a cofactor. The tissue distribution of KH activity in the rat suggested that the majority of active enzyme is located in liver and kidney. Detectable amounts were found in several other tissues, including brain which had low but significant levels of activity in every region assayed.     

The development of an immobilized lactate oxidase system for lactic acid analysis

An enzyme designated as lactate oxidase was purified from Acetobacter peroxydans by using the partition methods of separation. A DE-52 cellulose column was used for the primary purification of lactate oxidase, and the purified enzyme was covalently bound to a porous cellulose bead matrix in which benzoquinone was used as the coupling reagent. The physicochemical properties of the native and immobilized enzymes were determined including molecular weight, cofactor requirements, and optimal reaction conditions. Lactate oxidase was shown not to be subject to product inhibition, and to require Mg(2+) as a metal cofactor. Analysis of an immobilized lactate oxidase packed-bed reactor indicated that this system may not be subject to internal diffusional limitations. Molecular oxygen appeared to be a cosubstrate of the enzyme, and a reaction mechanism was postulated to predict the kinetic behavior of the immobilized reactor system. Applications of the immobilized lactate oxidase reactor for the pulse-flow analysis of lactic acid in whole milk and in a yeast fermentation system were considered.     

Identification of a molybdopterin-containing molybdenum cofactor in xanthine dehydrogenase from Pseudomonas aeruginosa.

Xanthine dehydrogenase has been purified from Pseudomonas aeruginosa cultured on a rich medium and induced with hypoxanthine. The enzyme was shown to contain FAD, iron sulfur centers and a molybdenum cofactor as prosthetic groups. Analysis of the molybdenum cofactor in this enzyme has revealed that the cofactor contains molybdopterin (MPT) rather than molybdopterin guanine dinucleotide or molybdopterin cytosine dinucleotide which have previously been identified in a number of molybdoenzymes of bacterial origin. The pterin cofactor in P.aeruginosa xanthine dehydrogenase was alkylated and the resulting product was identified as dicarboxamidomethyl molybdopterin. In addition, the pterin released from the enzyme by denaturation with guanidine-HCl was found to chromatograph on Sephadex G-15 with an apparent molecular weight of 350. These results document the first example of a bacterial enzyme with a molybdenum cofactor comprising molybdopterin and the metal only.     

Aclacinomycin 10-hydroxylase is a novel substrate-assisted hydroxylase requiring S-adenosyl-L-methionine as cofactor

Aclacinomycin 10-hydroxylase is a methyltransferase homologue that catalyzes a S-adenosyl-L-methionine (AdoMet)-dependent hydroxylation of the C-10 carbon atom of 15-demethoxy-epsilon-rhodomycin, a step in the biosynthesis of the polyketide antibiotic beta-rhodomycin. S-Adenosyl-L-homocysteine is an inhibitor of the enzyme, whereas the AdoMet analogue sinefungin can act as cofactor, indicating that a positive charge is required for catalysis. 18O2 experiments show that the hydroxyl group is derived from molecular oxygen. The reaction further requires thiol reagents such as glutathione or dithiothreitol. Incubation of the enzyme with substrate in the absence of reductant leads to the accumulation of an intermediate with a molecular mass consistent with a perhydroxy compound. This intermediate is turned into product upon addition of glutathione. The crystal structure of an abortive enzyme-AdoMet product ternary complex reveals large conformational changes consisting of a domain rotation leading to active site closure upon binding of the anthracycline ligand. The data suggest a mechanism where decarboxylation of the substrate results in the formation of a carbanion intermediate, which is stabilized by resonance through the aromatic ring system of the anthracycline substrate. The delocalization of the electrons is facilitated by the positive charge of the cofactor AdoMet. The activation of oxygen and formation of a hydroxyperoxide intermediate occurs in a manner similar to that observed in flavoenzymes. Aclacinomycin-10-hydroxylase is the first example of a AdoMet-dependent hydroxylation reaction, a novel function for this cofactor. The enzyme lacks methyltransferase activity due to the positioning of the AdoMet methyl group unfavorable for a SN2-type methyl transfer to the substrate.     

Molybdopterin cofactor from Methanobacterium formicicum formate dehydrogenase.

The molybdopterin cofactor from the formate dehydrogenase of Methanobacterium formicicum was studied. The cofactor was released by guanidine denaturation of homogeneous enzyme, which also released greater than 80% of the molybdenum present in the enzyme. The anoxically isolated cofactor was nonfluorescent, but after exposure to air it fluoresced with spectra similar to those of described molybdopterin cofactors. Aerobic release from acid-denatured formate dehydrogenase in the presence of I2 and potassium iodide produced a mixture of fluorescent products. Alkaline permanganate oxidation of the mixture yielded pterin-6-carboxylic acid as the only detectable fluorescent product. The results showed that the cofactor from formate dehydrogenase contained a pterin nucleus with a 6-alkyl side chain of unknown structure. Covalently bound phosphate was also present. The isolated cofactor was unable to complement the cofactor-deficient nitrate reductase of the Neurospora crassa nit-1 mutant.     

The structural basis of the recognition of phenylalanine and pterin cofactors by phenylalanine hydroxylase: implications for the catalytic mechanism

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin and non-heme iron-dependent enzyme that hydroxylates L-Phe to l-Tyr using molecular oxygen as additional substrate. A dysfunction of this enzyme leads to phenylketonuria (PKU). The conformation and distances to the catalytic iron of both L-Phe and the cofactor analogue L-erythro-7,8-dihydrobiopterin (BH2) simultaneously bound to recombinant human PAH have been estimated by (1)H NMR. The resulting bound conformers of both ligands have been fitted into the crystal structure of the catalytic domain by molecular docking. In the docked structure L-Phe binds to the enzyme through interactions with Arg270, Ser349 and Trp326. The mode of coordination of Glu330 to the iron moiety seems to determine the amino acid substrate specificity in PAH and in the homologous enzyme tyrosine hydroxylase. The pterin ring of BH2 pi-stacks with Phe254, and the N3 and the amine group at C2 hydrogen bond with the carboxylic group of Glu286. The ring also establishes specific contacts with His264 and Leu249. The distance between the O4 atom of BH2 and the iron (2.6(+/-0.3) A) is compatible with coordination, a finding that is important for the understanding of the mechanism of the enzyme. The hydroxyl groups in the side-chain at C6 hydrogen bond with the carbonyl group of Ala322 and the hydroxyl group of Ser251, an interaction that seems to have implications for the regulation of the enzyme by substrate and cofactor. Some frequent mutations causing PKU are located at residues involved in substrate and cofactor binding. The sites for hydroxylation, C4 in L-Phe and C4a in the pterin are located at a distance of 4.2 and 4.3 A from the iron moiety, respectively, and at 6.3 A from each other. These distances are adequate for the intercalation of iron-coordinated molecular oxygen, in agreement with a mechanistic role of the iron moiety both in the binding and activation of dioxygen and in the hydroxylation reaction.     

The tyrosine-dependent oxidation of tetrahydropterins by lysolecithin-activated rat liver phenylalanine hydroxylase

In the presence of tyrosine, phenylalanine hydroxylase, which has been activated with lysolecithin, catalyzes the oxidation of tetrahydrobiopterin at a rate 10-20% that of the parallel reaction with phenylalanine. Unlike the reaction with phenylalanine, there is no net concomitant hydroxylation of tyrosine, although the amino acid is still a necessary component. Tyrosine appears to form an abortive complex with the activated enzyme, the pterin cofactor and molecular oxygen. The Km for tetrahydrobiopterin is identical for the reactions with phenylalanine and tyrosine, whereas the Km for tyrosine is approximately 3 1/2 times greater than the Km for phenylalanine. The tyrosine-dependent oxidation of tetrahydrobiopterin proceeds at both pH 6.8 and 8.2 and shows a similar dependence on the pH as that of the physiological reaction. Tetrahydrobiopterin can be replaced by the artificial cofactor, 6-methyltetrahydropterin, in the tyrosine-dependent oxidation at both pH 6.8 and 8.2. As in the parallel reaction with phenylalanine, both the Km for the cofactor and the Km for the aromatic amino acid increase with this substitution.     

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

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

Trimethylamine oxidation in liver tissue is not catalyzed by a molybdenum cofactor-dependent enzyme

The oxidation of trimethylamine to trimethylamine N-oxide in animals is catalyzed by an enzyme which has not yet been fully characterized. The discovery that a bacterial enzyme catalyzing the reverse reaction, the reduction of trimethylamine N-oxide to trimethylamine, utilizes the molybdenum cofactor to carry out this function raised the possibility that trimethylamine oxidation may also be dependent on this cofactor. It was found, however, that liver tissue from tungsten-treated rats contained normal levels of trimethylamine oxidase. In addition, analysis of a urine sample from a patient with trimethylamine oxidase deficiency revealed the presence of normal levels of urothione, the degradation product of the molybdenum cofactor. These results suggest that trimethylamine oxidase is not a molybdoenzyme and that oxidation of trimethylamine proceeds by a mechanism which differs considerably from a simple reversal of trimethylamine N-oxide reduction.     

Initial steps of catabolism of trihydroxybenzenes in Pelobacter acidigallici

The initial steps of the anaerobic degradation of trihydroxylated aromatic monomers were investigated in a strain (AG2) isolated on gallic acid and identified as Pelobacter acidigallici. Kinetic studies showed that strain AG2 fermented gallic acid into acetate with a transient accumulation of pyrogallol and phloroglucinol in the medium. In addition phloroglucinol was produced from all other trihydroxylated aromatic monomers and pyrogallol from 2,3,4-trihydroxybenzoate. Although protocatechuate did not support growth of the organism, it was partially decarboxylated by resting cells of strain AG2. Cell free extract of strain AG2 catalysed the oxidation of NADPH in presence of resorcinol, 2,4,6-trihydroxybenzoate and phloroglucinol. However, comparison of activities indicated that the latter was the true physiological electron acceptor. Phloroglucinol and its reduction product dihydrophloroglucinol appeared thus to play a key role in metabolism of trihydroxybenzenes and a unified pathway, involving a decarboxylation of trihydroxybenzoates, a para transhydroxylation of pyrogallol into phloroglucinol and the formation of dihydrophloroglucinol, was proposed.     

Metabolism of gallate and phloroglucinol in Eubacterium oxidoreducens via 3-hydroxy-5-oxohexanoate

The pathway for the anaerobic catabolism of gallic acid by Eubacterium oxidoreducans was studied by using both in vivo and cell-free systems. Cells grown with gallate and crotonate, but with no formate or H2, excreted pyrogallol and phloroglucinol into the medium. Gallate was decarboxylated by crude cell extracts, with pyrogallol as the only detectable product. Whole cells converted pyrogallol to phloroglucinol. A phloroglucinol reductase catalyzed the conversion of phloroglucinol to dihydrophloroglucinol when NADPH was used as the source of electrons. Both formate dehydrogenase (EC 1.2.1.43) and hydrogenase (EC 1.18.99.1) were present in cell extracts of gallate-formate-grown cells. These two enzymes were both NADP linked. Since either H2 or formate is required for cell growth with gallate or phloroglucinol, these results suggest that the oxidation of the reduced substrate may be indirectly linked to the reduction of phloroglucinol. A dihydrophloroglucinol hydrolase was present, which hydrolyzed dihydrophloroglucinol to 3-hydroxy-5-oxohexanoate. This six-carbon ring cleavage product then presumably can be broken down by a series of reactions similar to beta-oxidation. These reactions cleaved the six-carbon acid to 3-hydroxybutyryl-coenzyme A yielding acetate and butyrate as end products. A number of key enzymes involved in beta-oxidation and substrate-level phosphorylation were demonstrated in cell extracts.     

Mechanism of oxygen activation by tyrosine hydroxylase

The mechanism by which the tetrahydropterin-requiring enzyme tyrosine hydroxylase (TH) activates dioxygen for substrate hydroxylation was explored. TH contains one ferrous iron per subunit and catalyzes the conversion of its tetrahydropterin cofactor to a 4a-carbinolamine concomitant with substrate hydroxylation. These results are in accord with shared mechanisms of oxygen activation by TH and the more commonly studied tetrahydropterin-dependent enzyme phenylalanine hydroxylase (PAH) and strongly suggest that a peroxytetrahydropterin is the hydroxylating species generated during TH turnover. In addition, TH can also utilize H2O2 as a cofactor for substrate hydroxylation, a result not previously established for PAH. A detailed mechanism for the reaction is proposed. While the overall pattern of tetrahydropterin-dependent oxygen activation by TH and PAH is similar, the H2O2-dependent hydroxylation performed by TH provides an indication that subtle differences in the Fe ligand field exist between the two enzymes. The mechanistic ramifications of these results are briefly discussed.     

Quantitative transfer of the molybdenum cofactor from xanthine oxidase and from sulphite oxidase to the deficient enzyme of the nit-1 mutant of Neurospora crassa to yield active nitrate reductase

An assay method is described for measurement of absolute concentrations of the molybdenum cofactor, based on complementation of the defective nitrate reductase ('apo nitrate reductase') in extracts of the nit-1 mutant of Neurospora crassa. A number of alternative methods are described for preparing, anaerobically, molybdenum-cofactor-containing solutions from sulphite oxidase, xanthine oxidase and desulpho xanthine oxidase. For assay, these were mixed with an excess of extract of the nit-1 mutant, incubated for 24 h at 3.5 degrees C then assayed for NADPH:nitrate reductase activity. In all cases, the specific activity of the molybdenum cofactor, expressed as mumol of NO2-formed/min per ng-atom of Mo added from the denatured molybdoenzyme , was 25 +/- 4, a value that agrees with the known catalytic activity of the nitrate reductase of wild-type Neurospora crassa. This indicates that, under our conditions, there was quantitative transfer of the molybdenum cofactor from denatured molybdoenzyme to yield fully active nitrate reductase. Comparable cofactor assay methods of previous workers, apparently indicating transfer efficiencies of at best a few per cent, have never excluded satisfactorily the possibility that cofactor activity arose, not from stoichiometric constituents of the molybdoenzymes , but from contaminants. The following factors were investigated separately in developing the assay:the efficiency of extraction of the cofactor from the original enzyme, the efficiency of the complementation reaction between cofactor and apo nitrate reductase, and the assay of the resultant nitrate reductase, which must be carried out under non-inhibitory conditions. Though the cofactor is unstable in air (t1/2 about 15 min at 3.5 degrees C), it is stable when kept anaerobic in the presence of sodium dithionite, in aqueous solution or in dimethyl sulphoxide (activity lost at the rate of about 3%/24 h at 20-25 degrees C). Studies of stabilities, and investigations of the effect of added molybdate on the assay, permit conclusions to be drawn about the ligation of molybdenum to the cofactor and about steps in incorporation of the cofactor into the apoenzyme. Though the development of nitrate reductase activity is slow at 3.5 degrees C (t1/2 1.5-3 h) the complementation reaction may be carried out in high yield, aerobically. This is ascribed to rapid formation of an air-stable but catalytically inactive complex of the cofactor, as a precursor of the active nitrate reductase.(ABSTRACT TRUNCATED AT 400 WORDS)