Molybdenum Hydroxylases Research Articles

Hydralazine: A potent inhibitor of aldehyde oxidase activity in vitro and in vivo

The interaction of the vasodilator, hydralazine, with the molybdenum hydroxylases, aldehyde oxidase and xanthine oxidase has been investigated. A potent progressive inhibition of rabbit liver aldehyde oxidase, in the presence of substrate, by low concentrations of hydralazine (0.1–1 μM) was observed in vitro but no effect was seen with bovine milk xanthine oxidase. This activity was mirrored in vivo when levels of aldehyde oxidase were significantly decreased in rabbits administered hydralazine (10 mg/kg/day for seven days) whereas hepatic xanthine oxidase activity was unaltered by hydralazine treatment. Various metabolites of hydralazine were synthesized but found to be devoid of in vitro inhibitory activity. Aldehyde oxidase prepared from either guinea pig or baboon liver was inhibited in a similar way to that of rabbit liver.

Elevation of molybdenum hydroxylase levels in rabbit liver after ingestion of phthalazine or its hydroxylated metabolite

Oral administration of phthalazine (50 mg/kg/day) or 1-hydroxyphthalazine (10 mg/kg/day) to female rabbits caused an increase in the specific activity of the hepatic molybdenum hydroxylases aldehyde oxidase and xanthine oxidase, whereas no effect on microsomal cytochrome P-450 activity was observed. The rise in the specific activity of purified aldehyde oxidase fractions was accompanied by a similar increase in molybdenum content. A significant lowering of the K m value for phthalazine was demonstrated with enzyme from treated rabbits whereas K m values for structurally similar substrates such as isoquinoline were unchanged from control values. Iso-electric focusing of DEAE-cellulose fractions showed the presence of an additional band of activity indicating that genuine induction of aldehyde oxidase had occurred in rabbits treated with phthalazine or 1-hydroxyphthalazine.

Thermophilic Bacilli growing with carbon monoxide

Four strains of obligately thermophilic Bacilli capable of growing with carbon monoxide as a sole carbon and energy source were isolated from settling ponds of a sugar factory. Most of them could be identified as strains of Bacillus schlegelii on the basis of cell wall composition, DNA homology menaquinone and DNA base content. Growth with CO was very fast (t d =3 h) and was optimal at 65°C. No growth occurred below 50°C. As with the mesophilic carboxydotrophs, hydrogen plus carbon dioxide could also serve as autotrophic substrates. Growth of the isolates with CO depended on the presence of molybdenum in the growth medium. This suggested CO oxidase in the newly isolated Bacilli being a molybdenum hydroxylase similar to the enzymes from the mesophilic carboxydotrophs. Some data characterizing the CO-oxidizing activity in extracts of the thermophilic isolates are also provided.

Modelling the molybdenum centers of the molybdenum hydroxylases

Modelling the molybdenum centers of the molybdenum hydroxylases

Modeling the molybdenum sites of the molybdenum hydroxylases

Molybdenum hydroxylases are multicomponent enzymes which catalyze two electron oxidations of ▪ purines, aldehydes, formate and sulfite in animals and microorganisms [1]. In addition, eukaryotic nitrate reductase [2] and several as yet poorly characterized molybdenum containing enzymes have properties similar to those of the hydroxylases. Recent EPR and EXAFS investigation indicate the presence of a terminal oxo and a terminal sulfido group on Mo in oxidized (Mo(VI)) xanthine oxidase and xanthine dehydrogenase and two oxo groups in oxidized sulfite oxidase [1, 3]. In the reduced state (Mo(V), (IV)) the sulfido group appears to be converted to SH (xanthine oxidase, xanthine dehydrogenase) or one oxo to OH (sulfite oxidase) [3]. In addition, 2–3 Mo thiolate sulfur ligands are present for both oxidized and reduced enzymes [3]. One or more of the thiolate ligands may be located on a side chain of a reduced pterin proposed to be the cofactor common to all Mo hydroxylases [4]. The reduction potentials of the Mo centers of the hyroxylases have been determined and are found to differ considerably between enzymes (−0.355 V for the Mo(VI)/(V) couple in xanthine oxidase [5], 0.038 V in sulfite oxidase [6], e.g.). Recent model studies have concentrated on synthesis and structural characterization of dioxo-Mo(VI) complexes with N, S donor sets which mimic the EXAFS results [7] (Fig. 1a), on monomeric oxo-Mo(V) complexes having EPR parameters similar to those of the enzymes [8, 9] (Fig. 1b), and on oxo-Mo(VI), (V) and (IV) complexes which mimic the redox behavior of the enzymes [9, 10] (Fig. 1b). These results are briefly reviewed. Current work in this laboratory is directed towards the synthesis and characterization of dioxo-Mo(VI) complexes with sterically bulky bi-, tri- and tetradentate ligands which may be electrochemically or chemically reduced to monomeric Mo(V)(O)(OH) complexes: ▪ Normally, such reductions give oxo bridged Mo(V) dimers; the presence of bulky groups on the ligands, however, inhibits the dimerization. Representative ligands: ▪ Synthetic methods for the preparation of the complexes and their properties (IR, electronic and EPR spectra; electrochemistry) are reported. The relationships between EPR and electrochemical parameters and structures of the complexes are explored and the implications for the molybdenum hydroxylases are discussed. Current problems in modeling the molybdenum centers of the hydorxylases and possible directions for research toward the solutions of these problems are presented.

Modeling the molybdenum centers of the molybdenum hydroxylases

Modeling the molybdenum centers of the molybdenum hydroxylases

The fate of dibenz[b,f]-1,4-oxazepine (CR) in the rat. Part II. Metabolism in vitro.

CR (dibenz[b,f]-1,4-oxazepine) is metabolized by rat liver 105 000 g supernatant fractions by (a) ring opening and reduction to 2-amino-2'-hydroxymethyldiphenyl ether and (b) oxidation at C11 to give a cyclic lactam. Reaction (a) is NADPH-dependent, decreased by dialysis and methylene blue, whereas reaction (b) is heat-resistant, inactivated by dialysis, inhibited by CN-, p-chloromercuribenzoate, amytal and menadione, and stimulated by methylene blue, phenazine methosulphate and 2,6-dichlorophenol indophenol. Reaction (a) is similar to that of aldehyde reductases (E.C.1.1.1.2) and reaction (b) to that of molybdenum hydroxylases (E.C.1.2.3.1). Reaction (a) is also catalysed by an NADH-dependent enzyme in liver microsomes and subsequent hydroxylation of the lactam also occurs in this cell fraction. Some extrahepatic metabolism of CR occurs via the same routes in kidney, small intestine and lung, though the yield is limited. Digestive gland extract of Helix pomatia converts CR to its lactam in significant amounts. The metabolism of CR in vitro is similar to that predicted from observations in vivo.

Chemical and spectral properties of carbon monoxide: methylene blue oxidoreductase. The molybdenum-containing iron-sulfur flavoprotein from Pseudomonas carboxydovorans.

Carbon monoxide:methylene blue oxidoreductase, the key enzyme of CO-oxidation in energy metabolism of the carboxydobacterium Pseudomonas carboxydovorans, has been isolated in good yield and purity and found to contain FAD, molybdenum, iron, and labile sulfide in the ratio of 1:1:4:4. The enzyme is, therefore, a new molybdenum-containing iron-sulfur flavoprotein, exhibiting chemical and spectral properties quite similar to those of xanthine oxidase. Analytical data on the spectral characteristics of the enzyme in the oxidized and various reduced states are presented. Carbon monoxide:methylene blue oxidoreductase turned out to be photoreducible in the presence of EDTA and urea and was subject to reoxidation by air oxygen; no flavoprotein semiquinone was formed. Unphysiological electron acceptors, e.g. methylene blue, were used as oxidizing substrates whereas NAD or NADP turned out to be ineffective. Methylene blue reduction with CO was not affected by the presence of allopurinol, and carbon monoxide:methylene blue oxidoreductase was not able to catalyze the reduction of methylene blue with xanthine, adenine, or aldehydes. CO was the only reducing substrate used by the enzyme. Carbon monoxide:methylene blue oxidoreductase formed no sulfite adduct, and the reactivity with ferricyanide or cytochrome c was significant but slow. As known for other molybdenum hydroxylases, carbon monoxide:methylene blue oxidoreductase was rapidly inactivated by methanol, but the enzyme exhibited no ability to catalyze the oxidation of NADH with methylene blue, and NAD was not able to overcome methanol inhibition.

Molybdenum hydroxylases in Drosophila. II. Molybdenum cofactor in xanthine dehydrogenase, aldehyde oxidase and pyridoxal oxidase.

The molybdenum hydroxylases are a ubiquitous class of enzymes which contain molybdenum in association with a low molecular weight cofactor. Genetic evidence suggests that the Drosophila loci, ma--1, cin and lxd are concerned with this cofactor because mutants for any one of these loci simultaneously interrupt activity for two molybdenum hydroxylases, XDH and A0. A third enzyme activity, P0, is also absent in each of the three mutants but evidence classifying P0 as a molybdoenzyme has been lacking. This study utilizes the known tungsten sensitivity of molybdoenzymes to demonstrate directly that pyridoxal oxidase is also molybdoenzyme. The low molecular weight molybdenum cofactor is found to be severely reduced in extracts of the 1xd and cin mutants but ma--1 mutants have high levels of cofactor. A partially purified preparation of XDH crossreacting material from ma--1 was also shown to contain the molybdenum cofactor. These results, considered with data from other workers are taken to indicate that the functions of all three of the loci examined could be concerned with some aspect of cofactor biosynthesis.

Molybdenum hydroxylases in Drosophila

Molybdenum hydroxylases in Drosophila

Molybdenum-containing enzymes

Recent work on the molybdenum-containing enzymes has made it clear that they have many features in common. With the exception of nitrogenase, all those examined under suitable conditions give EPR signals from Mo(V). These include xanthine oxidase and closely related enzymes (the molybdenum-containing hydroxylases), sulphite oxidase and nitrate reductase. A variety of chemical modifications affect the properties of the metal in the molybdenum hydroxylases. However, in the normal reduced form of xanthine oxidase (which gives the EPR signal known as Rapid), redox potentials (at pH 8.2 and 25 °C) are Mo(VI)/Mo(V) −355 mV and Mo(V)/Mo(IV) −355 mV; substrates are believed to reduce the metal directly from Mo(VI) to Mo(IV). The EPR spectra of the enzymes show interaction with protons; this interaction is probably due to structures of the type Mo-NR-H. Though EPR can, in principle, give information on the ligand environment of the metal in the enzymes, work in this area is handicapped by a lack of well characterised mononuclear Mo(V) compounds suitable to serve as models.

Evidence for semiquinone-metal interaction in metal-flavoproteins

This chapter discusses the biological distribution and substrate-inhibitor specificity of molybdenum hydroxylases. Aldehyde oxidase and xanthine oxidase are molybdenum-containing enzymes that catalyze the oxidation of many aldehydes and nitrogen heterocycles. They have also been shown to catalyze, under in vitro conditions, a number of reductions. Although aldehyde oxidase is known to oxidize physiological compounds, such as N1-methylnicotinamide and pyridoxal, there does not yet appear to be a specific endogenous role for this enzyme. Xanthine oxidase is involved in endogenous purine catabolism, catalyzing the oxidation of hypoxanthine and xanthine to uric acid. This enzyme also has wide substrate specificity, and different functions have been assigned to it. One of the most plausible proposals is that both molybdenum hydroxylases may provide a biochemical barrier against ingested purines and pyrimidines, rendering them harmless. This is similar to the view put forward in a recent review on the role of the molybdenum hydroxylases as drug-metabolizing enzymes, except that the substrate specificity is not restricted to purines and pyrimidines.