Extraction and purification of molybdenum cofactor from milk xanthine oxidase

Molybdenum cofactor (mocofactor) is extracted efficiently, free of impurities and in high concentrations, by acid treatment of xanthine oxidase and subsequent incubation of the precipitate with phosphate buffer containing EDTA, molybdate and oxygen. It is suggested that cofactor is bound to the enzyme via hydrophobic forces as well as via an oxygen-sensitive mechanism. Upon extraction, the capability to complement the apo nitrate reductase of Neurospora crassa nit-1 can be conserved only in the total absence of oxygen. Cysteine and glutathione were shown to protect efficiently free mocofactor from oxidation. Two species of active mocofactor, probably a molybdoform and a demolybdoform, could be separated by means of reversed-phase HPLC with a mobile phase of 5 mM sodium citrate at a pH of 6.5. The mode of interaction between either of these species with thiol reagents is discussed.     

The oxidation product of molybdenum cofactor from milk xanthine oxidase

In extracts of acid treated molybdenum cofactor containing xanthine oxidase, fluorescence is maximally developed upon a three hours incubation. Analysis by means of reversed phase HPLC revealed the presence of several fluorescent compounds, the main one being a blue fluorescent compound with an emission maximum of 465 nm when maximal excited at 395 nm at a neutral pH. Definite proof is presented that this compound is the oxidation product of the molybdenum cofactor. The remaining fluorescent products are shown to be pterin-derivatives, yielding predominantly pterin-6-carboxylic acid upon permanganate oxidation. Purified oxidation product of molybdenum cofactor however, didn's yield a fluorescent derivative at all upon treatment with permanganate.     

EXAFS studies of the molybdenum center of xanthine oxidase.

EXAFS spectra associated with the K-absorption edge of molybdenum in the desulpho and functional forms of xanthine oxidase and some potential synthetic analogues have been obtained. These data indicate that the immediate environment of the molybdenum is different in the two forms of the enzyme and that desulpho xanthine oxidase resembles that in [MoO2(S2CNEt2)2] and [MoO2(ethylcysteine)2]. The cyanolysable sulphur atom of functional xanthine oxidase is suggested to be tightly bound to the molybdenum at a distance of less than or equal to 2.3 A.     

Magnetic coupling of the molybdenum and iron-sulphur centres in xanthine oxidase and xanthine dehydrogenases.

Magnetic interaction between molybdenum and one of the iron-sulphur centres in milk xanthine oxidase [Lowe, Lynden-Bell & Bray (1972) Biochem. J. 130, 239-249] was studied further, with particular reference to the newly discovered Mo(V) e.p.r.(electron-paramagnetic-resonance) signal, Resting II [Lowe, Barber, Pawlik & Bray (1976) Biochem. J. 155, 81-85]. E.p.r. measurements at 35GHz near to 4.2K showed that the interaction has the same sign at all molybdenum orientations and is ferromagnetic. The predicted splitting of the e.p.r. signal from the reduced iron-sulphur centre, Fe/S I, was observed, Providing positive identification of this as the other interacting species. Chemical modification of the molybdenum environment in xanthine oxidase can change the size of the interaction severalfold, but interaction always remains approximately isotropic. The interaction in turkey liver xanthine dehydrogenase is indistinguishable from that in the oxidase. However, a bacterial xanthine dehydrogenase with different iron-sulphur centres shows rather larger interaction. Guanidinium chloride disturbs the iron-sulphur centres of the oxidase, and when this occurs there is a parallel and relatively small change in the interaction. Removal of flavin from the molecule, or raising the pH to 12.0, changes the interaction slightly without affecting the chromophores themselves. It is concluded that the Fe/S I centre and the Mo are at least 1.0nm and probably nearer 2.5nm apart, and that the conformation of the protein between them is relatively stable up to pH 12.     

The structure of the molybdenum cofactor. Characterization of di-(carboxamidomethyl)molybdopterin from sulfite oxidase and xanthine oxidase

A di-(carboxamidomethyl) derivative of molybdopterin, the organic component of the molybdenum cofactor, has been prepared under conditions favoring retention of all of the structural features of the molecule. The specific radioactivity of [1-14C]iodoacetamide incorporated relative to the amount of phosphate indicated two alkylation sites per pterin. Energy-dispersive x-ray analysis of the derivative showed the presence of 2 sulfurs in the derivative. An exact mass corresponding to the molecular formula C14H18N7O5S2 was obtained for the MH+ ion of the alkylated, dephosphorylated compound by fast atom bombardment mass spectroscopy. 1H NMR spectra of the phosphorylated and dephosphorylated forms of alkylated molybdopterin, in conjunction with the other data, have provided strong corroboration of the validity of the proposed structure of molybdopterin (Johnson, J. L., and Rajagopalan, K. V. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6856-6860) as a 6-alkylpterin with a 4-carbon side chain containing an enedithiol on C-1' and C-2', a secondary alcohol on C-3', and a phosphorylated primary alcohol on C-4'. As isolated, the di-(carboxamido-methyl)molybdopterin was found to be a 5,6,7,8-tetrahydropterin.     

Proton electron-nuclear double-resonance spectra of molybdenum(V) in different reduced forms of xanthine oxidase

Electron-nuclear double-resonance (ENDOR) spectra of protons coupled to molybdenum(V) in reduced xanthine oxidase samples have been recorded. Under appropriate conditions these protons may be studied without interference from protons coupled to reduced iron-sulfur centers. Spectra have been obtained for the molybdenum(V) species known as Rapid, Slow, Inhibited, and Desulfo Inhibited. Resonances corresponding to at least nine protons or sets of protons are observed for all four species, with coupling constants in the range 0.08-4 MHz. Most of these protons do not exchange when 2H2O is used as solvent. Additional protons giving couplings up to 40 MHz are also detected. These correspond to EPR-detectable protons studied in earlier work. The strongly coupled protons may be replaced by 2H, through appropriate use of 2H2O or of 2H-substituted substrates, with consequent disappearance of the 1H resonances. In most cases the corresponding 2H ENDOR features have also been observed. The nature of the various coupled protons is briefly discussed. Results permit specific conclusions to be drawn about the structures of the Inhibited and Desulfo Inhibited species. In particular, the data indicate that the aldehyde residue of the Inhibited species has been oxidized and that the four protons derived from the ethylene glycol molecule in the Desulfo Inhibited species are not all equivalent. Recent assignments [Edmondson, D.E., & D'Ardenne, S.C. (1989) Biochemistry 28, 5924-5930] of the weakly coupled protons in the latter species appear not to be soundly based. The possibility of obtaining more detailed structural information from the spectra is briefly considered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron-nuclear double resonance spectroscopy of the desulfo-inhibited molybdenum(V) center in bovine milk xanthine oxidase

The "desulfo-inhibited" Mo(V) center of bovine milk xanthine oxidase has been investigated by electron-nuclear double resonance spectroscopy. Comparison of spectral data obtained from samples prepared with [1H4]ethylene glycol and with [2H4]ethylene glycol allowed assignment of proton resonance lines due to the methylene protons of the coordinated ethylene glycol (AH = 3.6 MHz). Deuterium resonance lines were observed with the deuterated sample (AD = 0.4 MHz). No spectral evidence was obtained for any weakly coupled nitrogen nuclei to the Mo center under a variety of conditions. Dissolution of the sample in D2O had little effect on the resonance lines centered about the proton Zeeman frequency, which shows they are not due to exchangeable protons and suggests the Mo center does not have contact with bulk solvent. A deuterium delta m = +/- 2 "forbidden" transition is observed at high radio-frequency power levels, which suggests either an exchangeable proton on a Mo ligand or a coordinated solvent. Weakly coupled, nonexchangeable proton lines are observed about the free proton frequency, which exhibit properties characteristic of alpha-protons. A number of arguments are presented to support the proposal that these protons originate from the C(1') and C(2') positions on the side chain of the molybdopterin cofactor.     

Resonance-enhanced Raman scattering from the molybdenum center of xanthine oxidase

The molybdenum center of xanthine oxidase has been examined by resonance Raman spectroscopy. Making use of the long-wavelength absorption of the reduced molybdenum center in complex with violapterin (the product of enzymic action of lumazine), resonance Raman spectra were obtained using laser excitation at 676.4 nm. Several internal vibrational modes of violapterin were found to be resonance-enhanced, and a number of bands in the 250-1100 cm-1 range, presumably arising from vibrational modes of the molybdenum coordination sphere, were also observed. Upon substitution of 18O for 16O in the molybdenum coordination sphere, bands at 1469, 853, 517, 325, and 276 cm-1 exhibited shifts of 5-12 cm-1 to lower energy. By analogy to previous vibrational studies of Mo-O-Mo and Mo-O-R model compounds, the 853, 517, and 276 cm-1 frequencies were judged consistent with a labeled Mo-O-R linkage of the complexed violapterin. More importantly, the relatively small frequency shifts observed in these and other vibrations upon incorporation of 18O are very similar to those observed by others for 18O-labeled phenol and metal-phenolate complexes (Pinchas, S., Sadeh, D., and Samuel, D. (1965) J. Phys. Chem. 69, 2259-2264; Pyrz, W. J., Rue, L. A., Stern, L. J., and Que, L. J., Jr. (1985) J. Am. Chem. Soc. 107, 614-620) that model iron-tyrosinate proteins. The relatively small isotope-induced frequency shifts in multiple bands are thus interpreted as resulting from vibrational mixing of internal coordinates involving the oxygen atom with internal ring motions of the aromatic species. No oxygen isotope-sensitive bands were observed in the 900-1100 cm-1 region where Mo = O stretching modes typically occur. In agreement with the conclusions of previous workers (Davis, M.D., Olson, J. S., and Palmer, G. (1982) J. Biol. Chem. 257, 14730-14737) we interpret our results to indicate that the absorption band appearing upon complexation of violapterin with the molybdenum center of reduced xanthine oxidase is a molybdenum-to-violapterin charge-transfer band. These results, as well as several other lines of evidence, are consistent with direct coordination of violapterin to molybdenum in the charge-transfer complex via the 7-hydroxyl group (i.e. the hydroxyl group introduced into substrate by the enzyme). The Mo=O stretching mode of the complex is presumably not resonance enhanced because it is orthogonal to the charge-transfer electronic transition, suggesting that coordination of violapterin is cis to the oxo group.(ABSTRACT TRUNCATED AT 400 WORDS)     

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)     

Numbers and exchangeability with water of oxygen-17 atoms coupled to molybdenum (V) in different reduced forms of xanthine oxidase

The effect of using [17O]water (24-50% enriched) as solvent on the Mo(V) electron paramagnetic resonance spectra of different reduced forms of xanthine oxidase has been investigated. All the Mo(V) signals are affected. Procedures are described, based on the use of difference spectral techniques, that facilitate interpretation of such spectra. The number of coupled oxygen atoms may be determined by estimation of the fraction of the spectrum that remains unchanged by the isotope at a known enrichment. For a species having two coupled oxygen atoms, the use of two different isotope enrichments permits elimination from the difference spectra of the contribution of the two singly substituted species. From the application of these methods, it is concluded that not only the strength of the hyperfine coupling of oxygen ligands of molybdenum but also their number and their exchangeability with the solvent vary from one reduced form of the enzyme to another. The inhibited species from active xanthine oxidase has been studied in the most detail. It has two weakly coupled oxygen atoms [A(17O)av = 0.1-0.2 mT] that do not exchange with the solvent. A cyclic structure is proposed for this species in which two oxygen ligands of molybdenum are bonded to the carbon of the formaldehyde or other alcohol or aldehyde molecule that reacted in producing the signal. Structures of the other signal-giving species from active xanthine oxidase (Very Rapid and Rapid types 1 and 2) are discussed, as is corresponding information on species from the desulfo enzyme and from sulfite oxidase.     

ABA3 is a molybdenum cofactor sulfurase required for activation of aldehyde oxidase and xanthine dehydrogenase in Arabidopsis thaliana.

The xanthine oxidase class of molybdenum enzyzmes requires a terminal sulfur ligand at the active site. It has been proposed that a special sulfurase catalyzes the insertion of this ligand thereby activating the enzymes. Previous analyses of mutants in plants indicated that the genetic locus aba3 is involved in this step leading to activation of the molybdenum enzymes aldehyde oxidase and xanthine dehydrogenase. Here we report the cloning of the aba3 gene from Arabidopsis thaliana and the biochemical characterization of the purified protein. ABA3 is a two-domain protein with a N-terminal NifS-like sulfurase domain and a C-terminal domain that might be involved in recognizing the target enzymes. Molecular analysis of three aba3 mutants identified mutations in both domains. ABA3 contains highly conserved binding motifs for pyridoxal phosphate and for a persulfide. The purified recombinant protein possesses a cysteine desulfurase activity, is yellow in color, and shows a NifS-like change in absorbance in the presence of L-cysteine. Pretreatment of ABA3 with a thiol-specific alkylating reagent inhibited its desulfurase activity. These data indicate a transsulfuration reaction similar to bacterial NifS. In a fully defined in vitro system, the purified protein was able to activate aldehyde oxidase by using L-cysteine as sulfur donor. Finally, we show that the expression of the aba3 gene is inducible by drought-stress.     

Hyperfine coupling of O to Mo(V) in the rapid epr signal of xanthine oxidase: Evidence for an oxygen ligand of the metal

The spectrum of the Rapid Mo(V) electron paramagnetic resonance signal from xanthine oxidase dissolved in ¹⁷O-enriched water is presented. Difference technqiues have been used to eliminate the ¹⁶O contribution. Clearly observed structure in the spectrum is attributed to moderately strong hyperfine coupling of one oxygen atom to molybdenum. Though complete interpretation of the spectrum has not been attempted, one component of A(¹⁷O) is about 1.6 mT. The possibility that the oxygen is present in a Mo---OH group, whose proton is the strongly-coupled proton of the Rapid signal, is discussed.     

The molybdenum cofactor biosynthetic protein Cnx1 complements molybdate-repairable mutants, transfers molybdenum to the metal binding pterin, and is associated with the cytoskeleton

Molybdenum (Mo) plays an essential role in the active site of all eukaryotic Mo-containing enzymes. In plants, Mo enzymes are important for nitrate assimilation, phytohormone synthesis, and purine catabolism. Mo is bound to a unique metal binding pterin (molybdopterin [MPT]), thereby forming the active Mo cofactor (Moco), which is highly conserved in eukaryotes, eubacteria, and archaebacteria. Here, we describe the function of the two-domain protein Cnx1 from Arabidopsis in the final step of Moco biosynthesis. Cnx1 is constitutively expressed in all organs and in plants grown on different nitrogen sources. Mo-repairable cnxA mutants from Nicotiana plumbaginifolia accumulate MPT and show altered Cnx1 expression. Transformation of cnxA mutants and the corresponding Arabidopsis chl-6 mutant with cnx1 cDNA resulted in functional reconstitution of their Moco deficiency. We also identified a point mutation in the Cnx1 E domain of Arabidopsis chl-6 that causes the molybdate-repairable phenotype. Recombinant Cnx1 protein is capable of synthesizing Moco. The G domain binds and activates MPT, whereas the E domain is essential for activating Mo. In addition, Cnx1 binds to the cytoskeleton in the same way that its mammalian homolog gephyrin does in neuronal cells, which suggests a hypothetical model for anchoring the Moco-synthetic machinery by Cnx1 in plant cells.     

Rapid type 2 molybdenum(V) electron-paramagnetic resonance signals from xanthine oxidase and the structure of the active centre of the enzyme.

Rapid type 2 molybdenum(V) e.p.r. signals from reduced functional xanthine oxidase have been further investigated. These signals, which show strong coupling of two protons to molybdenum, have been obtained under a variety of new conditions: specifically either at pH 8.2 in the presence of borate ions, or at pH 10.1--10.7 with or without various other additions. Parameters of the signals were obtained with the help of computer simulations. In at least some of these signals, the coupled protons must be located on the enzyme rather than on bound species. The relationship between type 1 and type 2 Rapid signals is discussed. They may represent geometrical isomers, or alternatively, hydroxyl uptake as a ligand of molybdenum may be involved in formation of type 2 species.