Mo Cofactor Research Articles

Identification of molybdoproteins in Clostridium pasteurianum.

Cells of Clostridium pasteurianum whose N source is switched from NH3 to N2 accumulate large amounts of molybdenum beginning 1.5 h before the detection of nitrogenase activity. Anaerobic multiphasic gel electrophoresis and anion-exchange chromatography were used to identify the molybdoproteins and molybdenum-containing components present in N2-fixing cells. In addition to molybdate, six distinct 99Mo-labeled species were detected, i.e., a membrane fragment, the MoFe protein of nitrogenase, formate dehydrogenase, a Mo "binding-storage" protein, a 30-kilodalton molybdoprotein, and a low-molecular-weight molybdenum species. Of these, the MoFe protein, formate dehydrogenase, and the Mo binding-storage protein were present in more than one zone because of complex formation with other proteins, partial denaturation, and variation in the amount of Mo bound to the protein, respectively. In addition to the six proteins, a soluble "free" Mo cofactor in the cytosol was detected by showing that it reconstituted nitrate reductase activity in crude extracts of the Neurospora crassa mutant nit-1.

The relationship of Mo, molybdopterin, and the cyanolyzable sulfur in the Mo cofactor

Reconstitution of the apoprotein of the molybdoenzyme nitrate reductase in extracts of the Neurospora crassa mutant nit-1 with molybdenum cofactor released by denaturation of purified molybdoenzymes is efficient in the absence of exogenous MoO 4 2− under defined conditions. Evidence is presented that this molybdate-independent reconstitution is due to transfer of intact Mo cofactor, a complex of Mo and molybdopterin (MPT), the organic constituent of the cofactor. This complex can be separated from denatured protein by gel filtration, and from excess MoO 4 2− by reverse-phase HPLC. Sulfite oxidase, native xanthine dehydrogenase, and cyanolyzed xanthine dehydrogenase are equipotent Mo cofactor donors. Other well-studied inactive forms of xanthine dehydrogenase are also shown to be good cofactor sources. Using xanthine dehydrogenase specifically radiolabeled in the cyanolyzable sulfur, it is shown that this terminal ligand of Mo is rapidly removed from Mo cofactor under the conditions used for reconstitution.

COMPLEMENTATION IN VARIANTS OF SULFITE OXIDASE DEFICIENCY

Hereditary disorders causing a deficiency of sulfite oxidase (SO) activity result in the accumulation of sulfite, sulfocysteine and thiosulfate and a clinical syndrome of seizures, developmental delay, and ectopia lentis. The defect may be in the structural enzyme protein (apoenzyme) of SO or due to the absence of its active molybdenum (Mo) cofactor, in which case Mo-dependent xanthine oxidase activity is also reduced. Study of fibroblast lines of one patient (A) with SO apoenzyme defect and 5 unrelated patients (B-F) with Mo cofactor deficiency revealed no detectable SO activity in any case. Complementation tests showed that SO activity was restored when Line B was cocultured with each of 5 other lines. No activity was detected in other pairwise combinations. Complementation occurred without heterokaryon formation, suggesting that the corrective factor may be a diffusible precursor of the cofactor. These findings confirm heterogeneity of SO deficiency; at least 3 variants can be distinguished. Line A has a mutation of the structural gene for SO apoenzyme which may have affected the binding of the heme or Mo cofactor but is without effect on its antigenicity. Among the cofactor deficient lines, Line B clearly has a defect different from and correctable by the other 4 lines. These 4 could have a common defect or non-complementing lesions in the biosynthetic pathway of the cofactor.

Mononuclear active sites of molybdoenzymes: chemical approaches to structure and reactivity

One of two general classes of molybdoenzymes contains mono— nuclear catalytic sites and effects oxidation or reduction of substrate x/x0 by oxygen atom transfer: X + (0)XO. The reaction LMoO2 + X —3LMo0 + X0 is well established in synthetic systems, and is frequently accompanied by dimerization of Mo(VI,IV) complexes to [LMo(V)0320. A general kinetic analysis allowing determination of oxotransfer rate constants in systems with p—oxo dimer formation is out— lined. EXAFS results for several enzymes indicate the minimal coordi— nation spheres Mo(VI)02(SR)2 and Mo(IV)0(SR)3 for oxidized and fully reduced forms, respecti'vely. In a synthetic approach to these sites, the ligand pyridine—2,6—bis(1 , 1—diphenylethanethiol) (L-N(SH)2) was prepared. Reaction with MoO 2(acac)2 afforded the 5-coordinate trigonal bipyramidal complex Mo02(L-NS2), which was converted to MoO(L-NS 2 (DMF) with Ph3 P in DMF • In this and other oxo-transfer reactions of these complexes, the gem—diphenyl groups sterically suppress dimerization to a OMo(V)-0-Mo(V)0 complex. This reaction is blocked in enzymes by protein structural constraints. MoO(L—NS2)(DMF) and Me2SO react to produce Mo02(L-NS2) and Me25. Substrate saturation kinetics are observed at sufficient Me2SO concentrations. The two reactions were coupled to generate a catalytic sulfoxide reduction/phosphine oxidation cycle. In a related reaction, d—biotin—d—sulfoxide is reduced by MoO(L-NS2)(DMF) to d-biotin. Inasmuch as d-biotin-d-sulfoxide reductase is a Mo cofactor—dependent enzyme, this reaction provides a meaningful model for an enzymatic oxo—transfer reaction • Kinetic data for various oxo—transfer reactions are presented and their relevance to enzymatic processes is discussed. INTRODUCTION Molybdenum—containing enzymes are of two types: nitrogenases (1—6), which catalyze the reduction of dinitrogen to ammonia, and oxo—transfer enzymes (2,3,7,8), which catalyze what is in effect the transfer of oxygen atoms from or to the substrates X/XO. The latter transformations may be written most simply as reaction [1] or, to emphasize the two—electron nature of the overall processes, as the half—reaction [2]. Representative molybdoenzymes of X+(O) xO [1] X+H20 ± XO+2H++2e [2] the oxo-transfer type are listed in Table 1 • For sulfite oxidase and xanthine oxidase/ dehydrogenase in particular, abundant evidence has demonstrated that a mononuclear Mo coordination unit is the catalytic site. In addition to this site, every enzyme that has been at least partially characterized contains one or more prosthetic groups (heme, flavin, Fe—S cluster) capable of electron storage and transfer. Both nitrogenase and oxo-transfer molybdoenzymes contain dissociable cofactors that are obligatory for catalytic activity but which, except for containing all the molybdenum in a given enzyme, are chemically unrelated. The cofactor of nitrogenase is a Mo—Fe—S cluster of incompletely defined composition and structure (9, 10). The cofactor (Mo—co) of oxo—transfer enzymes (11) is not yet well characterized but has been shown to contain a pterin nucleus carrying a potentially coordinating side chain. Certain leading results of the Duke group (12-14) bearing on the structure of Mo-co from several enzymes are schematically illustrated in Fig. 1. Denaturation of sulfite oxidase and xanthine dehydrogenase from chicken liver and Chlorella nitrate reductase with 6 M guanidine HC1 in the presence of KI and 12 led to dissociation of the same fluorescent species (form A). When denaturation was performed in boiling pH 2.5 solution, a different fluorescent species (form B) was released. The permanganate

Detection of nitrate reductase activity in nitrate reductase deficient mutants of barley ( Hordeum vulgare)

Nitrate reductase deficient mutants of barley Az12 and Az13 showed 40–50% in vivo enzyme activity as compared with the Steptoe non-mutant under strictly anaerobic conditions of assay. However, in vitro nitrate reductase activity could not be detected in the mutant extracts prepared by different methods. The extracts did not contain any inhibitory factor as judged by effect of in vitro nitrate reductase activity in the Steptoe non-mutant. Defective functioning of the Mo cofactor in the mutants was indicated by the fact that reduced benzyl viologen was ineffective as an electron donor. Incorporation of Mo-cofactor obtained from the Steptoe non-mutant into the mutant extract significantly reconstituted nitrate reductase activity. The reconstituted enzyme was NADH specific, indicating that coenzyme specificity is not altered in the mutants. Thus association of Mo-cofactor with the apoenzyme appears to be defective in the mutants.

De novo formation of the niaD gene directed protomer in the NADPH-nitrate reductase of Aspergillus nidulans

Evidence for the de novo formation of the niaD gene directed protomer in the multimeric NADPH-nitrate reductase from Aspergillus nidulans has been obtained by the use of structural gene ( niaD -421) and cofactor gene ( cnxH -388) mutant strains. These mutants were cultured as forced heterocaryons after a period in which they were labelled with L-[ 3 H]-histidine. Following nitrate induction with a 2 hr pulse of L-[ 14 C]-lysine, we traced the fate of constitutively formed niaD + protomers into active holoenzyme. Only enzyme synthesized de novo in response to nitrate had significant levels of the isotope available during induction. We concluded that the cnxH + gene directed Mo cofactor acts only on protomers formed de novo upon induction.

The molybdoenzyme system of Drosophila melanogaster. I. Sulfite oxidase: identification and properties. Expression of the enzyme in maroon-like (mal), low-xanthine dehydrogenase (lxd), and cinnamon (cin) flies.

Sulfite oxidase (sulfite: ferricytochrome c oxidoreductase; EC 1.8.2.1) has been detected in Drosophila melanogaster and some of its properties have been studied. In most respects this enzyme resembles the mammalian sulfite oxidases except for its molecular weight (148,000), which is somewhat higher than that of rat sulfite oxidase (116,000). Cytochrome c, potassium-ferricyanide, and oxygen can serve as electron acceptors in the oxidation of sulfite by the enzyme. Although definite evidence can be obtained only through the analysis of the pure enzyme, experiments involving tungstate feeding suggest that Drosophila sulfite oxidase is most probably a molybdoenzyme. Extracts of mal flies show normal levels of sulfite oxidase, whereas lxd flies have only 5-10% of the activity of wild type, and in cin flies the enzyme is apparently absent. While it is possible that the lxd and cin mutations are at some level responsible for the defective synthesis of a molybdenum-containing cofactor (supposed to be present in most molybdoenzymes), the evidence accumulated so far by several authors and the results of the present investigation argue against the involvement of a Mo cofactor in the multiple enzyme deficiencies observed in mal flies.

Reduction of cyclopropene as criterion of active-site homology between nitrogenase and its Fe–Mo cofactor

THE nitrogenase Fe–Mo cofactor (FeMo-co)1 is usually characterised by its unique ability to restore N2-fixing ability to extracts of certain nitrogenase-less mutants1,2 and by its spectral properties, some of which can be correlated with those of the FeMo protein itself3. The role of FeMo-co in nitrogenase function is not yet clear. Recently, it was reported that uncomplemented FeMo-co catalysed BH−4 reduction of C2H2 to C2H4 in a CO-inhibited, non-ATP-dependent process4. This was taken as evidence that FeMo-co is the active site for N2 reduction and binding in nitrogenase, although conversion of N2 to NH3 by cofactor-BH−4 mixtures was not observed4. C2H2 is readily reduced by many homogeneous and heterogeneous catalysts and can give only a single 2 e− reduction product, urging caution in exclusive reliance upon it as a model substrate5,6. The observation that nitrogenase catalyses reduction of cyclopropene to both propene and cyclopropane in a ∼2:1 ratio in vitro7 and also in vivo8 suggests a more demanding criterion8 for evaluation of FeMo-co catalysis, one which still avoids the difficult energetic requirements for N2 activation and reduction. This new criterion is strengthened by the fact that cyclopropene competitively inhibits N2 (ref. 9). We find that incubation of FeMo-co with a suitable ‘apoprotein’ extract yields a reconstituted FeMo-protein activity for reduction of cyclopropene to propene and cyclopropane. FeMo-co alone, however, only catalyses cyclopropane formation in the conditions described4 for C2H2 reduction. This result and other considerations suggest that active site homology between FeMo-co and nitrogenase has not yet been fully demonstrated on a function basis.