Proton translocation coupled to trimethylamine N-oxide reduction in anaerobically grown Escherichia coli.

Proton translocation coupled to trimethylamine N-oxide reduction was studied in Escherichia coli grown anaerobically in the presence of trimethylamine N-oxide. Rapid acidification of the medium was observed when trimethylamine N-oxide was added to anaerobic cell suspensions of E. coli K-10. Acidification was sensitive to the proton conductor 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847). No pH change was shown in a strain deficient in trimethylamine N-oxide reductase activity. The apparent H+/trimethylamine N-oxide ratio in cells oxidizing endogenous substrates was 3 to 4 g-ions of H+ translocated per mol of trimethylamine N-oxide added. The addition of trimethylamine N-oxide and formate to ethylenediaminetetraacetic acid-treated cell suspension caused fluorescence quenching of 3,3'-dipropylthiacarbocyanine [diS-C3-(5)], indicating the generation of membrane potential. These results indicate that the reduction of trimethylamine N-oxide in E. coli is catalyzed by an anaerobic electron transfer system, resulting in formation of a proton motive force. Trimethylamine N-oxide reductase activity and proton extrusion were also examined in chlorate-resistant mutants. Reduction of trimethylamine N-oxide occurred in chlC, chlG, and chlE mutants, whereas chlA, chlB, and chlD mutants, which are deficient in the molybdenum cofactor, could not reduce it. Protons were extruded in chlC and chlG mutants, but not in chlA, chlB, and chlD mutants. Trimethylamine N-oxide reductase activity in a chlD mutant was restored to the wild-type level by the addition of 100 microM molybdate to the growth medium, indicating that the same molybdenum cofactor as used by nitrate reductase is required for the trimethylamine N-oxide reductase system.     

Further characterization of trimethylamine N-oxide reductase from Escherichia coli, a molybdoprotein

Escherichia coli trimethylamine N-oxide (TMAO) reductase I, the major enzyme among inducible TMAO reductases, was purified to homogeneity by an improved method including heat treatment, ammonium sulfate precipitation, and chromatographies on Bio-Gel A-1.5m, DEAE-cellulose, and Reactive blue-agarose. The molecular weight was estimated by gel filtration to be approximately 200,000. A single subunit peptide with a molecular weight of 95,000 was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme contained 1.96 atoms of molybdenum, 0.96 atoms of iron, 1.52 atoms of zinc, and less than 0.4 atoms of acid-labile sulfur per molecular weight of 200,000. The absorption spectrum of the enzyme showed a peak at 278 nm and a shoulder at 288 nm, but no characteristic absorption was found from 350 to 700 nm. A fluorescent derivative of molybdenum cofactor was found when the enzyme was boiled with iodine in acidic solution; its fluorescence spectra were almost the same as those of the form A derivative of molybdopterin found in sulfite oxidase. The molybdenum cofactor released from heated TMAO reductase I reconstituted nitrate reductase in the extracts of Neurospora crassa mutant strain nit-1 lacking molybdenum cofactor. Thus, TMAO reductase I contains molybdopterin, which is a common constituent of some molybdenum-containing enzymes. Some kinetic properties were also determined.     

Coupled electron/proton transfer in complex flavoproteins: solvent kinetic isotope effect studies of electron transfer in xanthine oxidase and trimethylamine dehydrogenase

A solvent kinetic isotope effect study of electron transfer in two complex flavoproteins, xanthine oxidase and trimethylamine dehydrogenase, has been undertaken. With xanthine oxidase, electron transfer from the molybdenum center to the proximal iron-sulfur center of the enzyme occurs with a modest solvent kinetic isotope effect of 2.2, indicating that electron transfer out of the molybdenum center is at least partially coupled to deprotonation of the Mo(V) donor. A Marcus-type analysis yields a decay factor, beta, of 1.4 A(-1), indicating that, although the pyranopterin cofactor of the molybdenum center forms a nearly contiguous covalent bridge from the molybdenum atom to the proximal iron-sulfur center of the enzyme, it affords no exceptionally effective mode of electron transfer between the two centers. For trimethylamine dehydrogenase, rates of electron equilibration between the flavin and iron-sulfur center of the one-electron reduced enzyme have been determined, complementing previous studies of electron transfer in the two-electron reduced form. The results indicate a substantial solvent kinetic isotope effect of 10 +/- 4, consistent with a model for electron transfer that involves discrete protonation/deprotonation and electron transfer steps. This contrasts to the behavior seen with xanthine oxidase, and the basis for this difference is discussed in the context of the structures for the two proteins and the ionization properties of their flavin sites. With xanthine oxidase, a rationale is presented as to why it is desirable in certain cases that the physical layout of redox-active sites not be uniformly increasing in reduction potential in the direction of physiological electron transfer.     

Anaerobic induction of trimethylamine N-oxide reductase and cytochromes by dimethyl sulfoxide inEscherichia coli

Reduction of trimethylamine N-oxide is catalyzed by at least two enzymes inEscherichia coli: trimethylamine N-oxide reductase, which is anaerobically induced by trimethylamine N-oxide, and the constitutive enzyme dimethyl sulfoxide reductase. In this study, an increase in the specific activity of trimethylamine N-oxide reduction was observed in the anaerobic culture with dimethyl sulfoxide, but the specific activity of dimethyl sulfoxide reduction was not changed. The inducible enzyme trimethylamine N-oxide reductase was found in this culture. A marked expression of the structural genetorA for trimethylamine N-oxide reductase was also observed in atorA-lacZ gene fusion strain under anaerobic conditions with either trimethylamine N-oxide or dimethyl sulfoxide.l-Methionine sulfoxide and the N-oxides of adenosine, picolines, and nicotinamide slightly repressed expression of the gene. Membrane-boundb- andc-type cytochromes involved in the trimethylamine N-oxide reduction were also produced in a wild-type strain grown anaerobically with dimethyl sulfoxide. But thec-type cytochrome was not produced in thetorA-lacZ strain grown anaerobically with trimethylamine N-oxide or dimethyl sulfoxide; this suggests that there is a correlation between the expression oftorA and the synthesis of the cytochrome.     

Molybdenum cofactor requirement for in vitro activation of apo-molybdoenzymes of Escherichia coli

The apo-nitrate reductase precursor in an Escherichia coli chlB mutant preparation obtained following growth in the presence of tungstate is activated by incubation with protein FA and a heat-treated preparation from an E. coli crude extract. We show that the requirement for heat-treated E. coli crude extract can be fulfilled by material obtained from either of two heat-denatured purified E. coli molybdoenzymes, namely nitrate reductase or trimethylamine N-oxide reductase. Apo-trimethylamine N-oxide reductase precursor in the tungstate-grown chlB preparation can be activated in a similar manner with material from either heat-denatured molybdoenzyme. The active component in the denatured molybdoenzyme preparations is shown to be the molybdenum cofactor by Neurospora crassa nit1 molybdenum cofactor assay, size estimation and fluorimetric analysis. The direct demonstration of the requirement for molybdenum cofactor in the E. coli tungstate-grown chlB complementation system is an important step towards the molecular definition of the activation process and an understanding of the mechanism of cofactor acquisition during molybdoenzyme biosynthesis.     

Adenosine 3′:5′-cyclic monophosphate in young and senescent human fibroblasts during growth and stationary phase in vitro. Effects of prostaglandin E1 and of adrenaline

1. A mono-oxygenase, which oxidizes trimethylamine and other tertiary amines bearing methyl or ethyl groups, was partially purified sixfold from Pseudomonas aminovorans grown on trimethylamine as sole carbon source. 2. The preferred electron donor was NADPH. The enzyme had a pH optimum of 8.0-9.4 for trimethylamine oxidation, and 8.8-9.2 for dimethylamine oxidation. 3. The oxidation product of trimethylamine was shown to be trimethylamine N-oxide. Other tertiary amines were probably also converted into N-oxides. 4. The enzyme also oxidized secondary amines. 5. The oxidation of trimethylamine was only slightly inhibited by CO and not at all by KCN or proadifen hydrochloride (SKF 525-A), but was inhibited by trimethylsulphonium chloride, tetramethylammonium chloride, 2,4-dichloro-6-phenylphenoxyethylamine (Lilly 53325) and its NN-diethyl derivative (Lilly 18947). 6. The oxidation of dimethylamine showed a similar response to inhibitors and a parallel loss in activity on heating at 35 degrees C. 7. The activities of the trimethylamine mono-oxygenase, trimethylamine N-oxide demethylase and the secondary-amine mono-oxygenase increased severalfold during adaptation of succinate-grown bacteria to growth on trimethylamine, and the trimethylamine mono-oxygenase was the first enzyme to show an increase in activity. It is concluded that all three enzymes are involved in growth on trimethylamine by this organism.     

The Active Components of Methyl Viologen-Nitrate Reductase in Xanthine Oxidase

The components of the active molybdenum cofactor in xanthine oxidase was found. The molybdenum cofactor is responsible for the enzymatic activity of the methyl viologen-nitrate reduction. The inactivation of the methyl viologen-nitrate reductase by cyanide is accompanied by the extraction of sulfur from the enzyme. Cyanide inactivated enzyme can be reactivated by incubation with Na2S. The results suggest that the active site of the methyl viologen-nitrate reductase contains an atom of active sulfur which does not originate from the acid labile sulfur of the Fe/S cluster, neither originate from the organic sulfur of the cysteine residue, nor from the sulfur of persulfide. It is probably another type of inorganic sulfur near the molybdenum atoms, The flavin-free xanthine oxidase may be loss entirely its oxidation activity of xanthine to uric acid. In contrast, the activity of the methyl viologen-nitrate reductase is nearly completly insensitive to the flavinfree treatment. Studies on the Fe-free xanthine oxidase, obtained by metal-binding agent phenanthroline and by acid treatment, revealed Fe (in xanthine oxidase it is the Fe of the Fe/S cluster) is also one of the active conponents, functioning in the methyl viologen-nitrate reductase, besides molybdenum.     

Molybdenum cofactor: a compound in the in vitro activation of both nitrate reductase and trimethylamine-N-oxide reductase activities in Escherichia coli K12

Nitrate reductase (nitrite: (acceptor) oxidoreductase, EC 1.7.99.4) and trimethylamine N-oxide reductase (NADH : trimethylamine-N-oxide oxidoreductase, EC 1.6.6.9) activities were reconstituted by incubation of the association factor FA (the putative product of the chlB gene) with the soluble extract of the chlB mutant grown anaerobically in the presence of trimethylamine N-oxide. When soluble extracts of the chlB mutant grown on 10 mM sodium tungstate, a molybdenum competitor, were used in complementation systems, no enzymatic reactivation was observed. Heated extracts of the parental strain 541 were shown to contain a thermoresistant molybdenum cofactor by their ability to reactivate NADPH-nitrate reductase activity in the nit1 mutant of Neurospora crassa. By complementation of parental strain heated extract with association factor FA and soluble extract of the chlB mutant grown in the presence of sodium tungstate, we were able to show for the first time that the molybdenum cofactor is an activator common to the in vitro reconstitution of both nitrate reductase and trimethylamine-N-oxide reductase activities.     

Oxidation of molybdopterin in sulfite oxidase by ferricyanide. Effect on electron transfer activities

The attenuation of the sulfite:cytochrome c activity of sulfite oxidase upon treatment with ferricyanide was demonstrated to be the result of oxidation of the pterin ring of the molybdenum cofactor in the enzyme. Oxidation of molybdopterin (MPT) was detected in several ways. Ferricyanide treatment not only abolished the ability of sulfite oxidase to serve as a source of MPT to reconstitute the aponitrate reductase in extracts of the Neurospora crassa mutant nit-1 but also eliminated the ability of sulfite oxidase to reduce dichlorobenzenoneindophenol after anaerobic denaturation. Additionally, the absorption spectrum of anaerobically denatured ferricyanide-treated molybdenum fragment of rat liver sulfite oxidase was typical of fully oxidized pterins. Ferricyanide treatment had no effect on the protein of sulfite oxidase or on the sulfhydryl-containing side chain of MPT. Quantitation of the ferricyanide reaction showed that 2 mol of ferricyanide were reduced per mol of MPT oxidized, yielding a fully oxidized pterin. These results corroborate the previously reported conclusion that the native state of reduction of MPT in sulfite oxidase is at the dihydro level (Gardlik, S., and Rajagopalan, K.V. (1990) J. Biol. Chem. 265, 13047-13054). As a result of oxidation of the pterin ring, the affinity of MPT for molybdenum is decreased, leading to eventual loss of molybdenum. Because the loss of molybdenum is slow, a population of sulfite oxidase molecules can exist in which molybdenum is complexed to oxidized MPT. These molecules retain sulfite:O2 activity, a function apparently dependent solely on the molybdenum-thiolate complex, yet have greatly decreased sulfite:cytochrome c activity, a function requiring heme as well as the molybdenum center of holoenzyme. These observations suggest that the pterin ring of MPT participates in enzyme function, possibly in electron transfer, directly in catalysis, or by controlling the oxidation/reduction potential of molybdenum.     

Quantitation of molybdopterin oxidation product in wild-type and molybdenum cofactor deficient mutants of Chlamydomonas reinhardtii.

A simple and reliable procedure of oxidation of molybdenum cofactor (MoCo) from molybdoenzymes by autoclaving samples at 120 degrees C for 20 min yielded a single predominant fluorescent species that could be quantitatively determined by reverse phase high performance liquid chromatography. This method allowed detection and quantitation of molybdopterin in cell-free extracts of the green alga Chlamydomonas reinhardtii. The MoCo oxidation product from C. reinhardtii has the same chromatographic and spectral properties as that of milk xanthine oxidase and chicken liver sulfite oxidase. The oxidized species was also detected in molybdenum cofactor mutants of Chlamydomonas reinhardtii defective at the nit-3, nit-4, nit-5/nit-6 and nit-7 loci, which strongly suggests that active molybdenum cofactor itself is not directly involved in the control of its own biosynthetic pathway.     

Anaerobic growth of Escherichia coli on formate by reduction of nitrate, fumarate, and trimethylamine N-oxide.

Anaerobic growth of E. coli, strain K-10, depending on formate oxidation by nitrate, fumarate, and trimethylamine N-oxide was followed in a medium containing peptone. The presence of formate and peptone was indispensable for growth with fumarate and trimethylamine N-oxide reduction. While there was no growth in the absence of acceptor, growth was observed in the absence of formate by nitrate reduction though not as much as under aerobic conditions. Per mole consumed formate equimolar succinate or trimethylamine was formed, but 1.2 mole of nitrate was produced, probably depending partly on peptone oxidation. The molar growth yield on formate was found to be 6.5, 7.6, and 7.0 g cells/mole depending on the reduction of nitrate, fumarate, and trimethylamine N-oxide, respectively, suggesting the formation of one mole ATP coupled to the anaerobic electron transfers from formate.     

The isolation of demolybdo xanthine oxidase from bovine milk.

It was deduced many years ago from indirect evidence that demolybdo xanthine oxidase is present in normal bovine milk. This has now been confirmed by isolation of this enzyme form by a method based on the folate-gel affinity-chromatography procedure described Nishino & Tsushima [(1986) J. Biol. Chem. 261, 11242-11246]. Enzymic and spectroscopic properties of demolybdo xanthine oxidase, which retains flavin and iron-sulphur centres, are generally in accordance with expectations. Like the normal enzyme, it yields on denaturation material fluorescing at 460 nm. Molybdenum cofactor activity measured by the Neurospora crassa nit-1 assay in the presence of added molybdate was 33% of that of the normal enzyme. The absorption spectrum in the near-u.v. region differs slightly, but significantly, from that of the active and desulpho forms of the enzyme. It is concluded that the molybdenum cofactor site contains a pterin-like material not identical with that in the normal enzyme. The significance of the occurrence of demolybdo xanthine oxidase in milk is discussed, and evidence in the literature for demolybdo forms of other molybdoenzymes is briefly reviewed. Additional studies on the use of the affinity procedure for large-scale preparation of high-activity xanthine oxidase are described. In agreement with our ability to isolate the demolybdo enzyme, the procedure appears less effective in eliminating the demolybdo than the desulpho enzyme.     

Biology of the molybdenum cofactor

The transition element molybdenum (Mo) is an essential micronutrient for plants where it is needed as a catalytically active metal during enzyme catalysis. Four plant enzymes depend on molybdenum: nitrate reductase, sulphite oxidase, xanthine dehydrogenase, and aldehyde oxidase. However, in order to gain biological activity and fulfil its function in enzymes, molybdenum has to be complexed by a pterin compound thus forming the molybdenum cofactor. In this article, the path of molybdenum from its uptake into the cell, via formation of the molybdenum cofactor and its storage, to the final modification of the molybdenum cofactor and its insertion into apo-metalloenzymes will be reviewed.     

31P ENDOR studies of xanthine oxidase: coupling of phosphorus of the pterin cofactor to molybdenum (V).

31P ENDOR spectra are described for three different molybdenum(V) species in reduced xanthine oxidase samples. The spectra were not affected by removing the FAD from the enzyme, implying that this is located at some distance from molybdenum. Furthermore, in confirmation of the work of J. L. Johnson, R. E. London, and K. V. Rajagopalan [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6493-6497], NMR and chemical analysis of the phosphate content of highly purified xanthine oxidase showed there are only three phosphate residues per subunit of the enzyme. It is concluded that the ENDOR features are due to hyperfine coupling of the phosphate group of the pterin cofactor to the molybdenum atom. Evaluation of the dipolar component of the coupling has permitted estimation of the molybdenum-phosphorus distances as 7-12 A. This implies that the cofactor is in an extended conformation in the enzyme molecule. Less detailed 31P ENDOR data on sulfite oxidase are consistent with a similar conformation for the cofactor in this enzyme.     

Molybdenum cofactor deficiency in a patient previously characterized as deficient in sulfite oxidase

The metabolic status of a patient previously characterized as deficient in sulfite oxidase was reexamined applying new methodology which has been developed to distinguish between a defect specific to the sulfite oxidase protein and sulfite oxidase deficiency which arises as a result of molybdenum cofactor deficiency. Urothione, the metabolic degradation product of the molybdenum cofactor, was undetectable in urine samples from the patient. Analysis of molybdenum cofactor levels in fibroblasts by monitoring reconstitution of apo nitrate reductase in extracts of the Neurospora crassa mutant nit-1 revealed that cells from the patient were severely depleted. Quantitation of urinary oxypurines showed that hypoxanthine and xanthine were highly elevated while uric acid remained in the normal range. These results were interpreted to indicate a severe but incomplete deficiency of the molybdenum cofactor. The presence of very low levels of active cofactor, supporting the synthesis of low levels of active sulfite oxidase and xanthine dehydrogenase, could explain the metabolic patterns of sulfur and purine products and the relatively mild clinical symptoms in this individual.     

The peak height ratio of S-sulfonated transthyretin and other oxidized isoforms as a marker for molybdenum cofactor deficiency,measured by electrospray ionization mass spectrometry

Molybdenum cofactor deficiency is a fatal neurological disorder, which follows an autosomal-recessive trait and is characterized by combined deficiency of the enzyme, sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase. Early detection of molybdenum cofactor-deficient patients is essential for their proper care and genetic counseling of families at risk. We demonstrate the use of S-sulfonated transthyretin (TTR) as a marker for molybdenum cofactor deficiency. Plasma or sera obtained from 4 patients with molybdenum cofactor deficiency and 57 controls were studied by electrospray ionization mass spectrometry (ESIMS) following selective enrichment of TTR by immunoprecipitation using protein G/A agarose. The data obtained from molybdenum cofactor deficiency samples indicated a strong increase in the peak height of S-sulfonated TTR. A more significant difference was revealed if the peak height ratio of S-sulfonated TTR and the sum of the other oxidized TTR were determined. By accurate determination of the ratio, the samples of molybdenum cofactor deficiency patients could clearly be distinguished from controls without molybdenum cofactor deficiency.     

Studies on the interaction of NADPH with Rhodobacter sphaeroides biotin sulfoxide reductase

Rhodobacter sphaeroides biotin sulfoxide reductase (BSOR) contains the bis(molybdopterin guanine dinucleotide)molybdenum cofactor and catalyzes the reduction of D-biotin-D-sulfoxide to biotin. This protein is the only member of the dimethyl sulfoxide reductase family of molybdopterin enzymes that utilizes NADPH as the direct electron donor to the catalytic Mo center. Kinetic studies using stopped-flow spectrophotometry indicate that BSOR reduction by NADPH (>1000 s(-1)) is faster than steady-state turnover (440 s(-1)) and has shown that BSOR reduction occurs in concert with NADPH oxidation with no indication of a Mo(V) intermediate species. Because no crystallographic structure is currently available for BSOR, a protein structure was modeled using the structures for R. sphaeroides dimethyl sulfoxide reductase, Rhodobacter capsulatus dimethyl sulfoxide reductase, and Shewanella massilia trimethylamine N-oxide reductase as the templates. A potential NADPH-binding site was identified and tested by site-directed mutagenesis of residues within the area. Mutation of Arg137 or Asp136 reduced the ability of NADPH to serve as the electron donor to BSOR, indicating that the NADPH-binding site in BSOR is located in the active-site funnel of the putative structure where it can directly reduce the Mo center. Along with kinetic and spectroscopic data, the location of this binding site supports a direct hydride transfer mechanism for NADPH reduction of BSOR.     

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.     

Formation of thieno[3,2-g]pterines from the molybdenum cofactor

A fluorescent oxidation product of the molybdenum cofactor was isolated from Escherichia coli nitrate reductase (EC 1.9.6.1) and bovine milk xanthine oxidase (EC 1.2.3.2), which showed a visible absorption band at 395 nm and was dephosphorylated by alkaline phosphatase but not by phosphodiesterase I. The dephosphorylated species was oxidized by periodate to thieno[3,2-g]pterin-2-carbaldehyde which was quantitatively converted to thieno[3,2-g]pterin-2-carboxylic acid by subsequent treatment with Ag2O in 2 N NaOH. These results indicate that the oxidation product of the molybdenum cofactor is a thieno[3,2-g]pterin derivative with an unidentified side chain in the 2 position.     

The molybdoenzyme formylmethanofuran dehydrogenase from Methanosarcina barkeri contains a pterin cofactor

Recently formylmethanofuran dehydrogenase from the archaebacterium Methanosarcina barkeri has been shown to be a novel molybdo-iron-sulfur protein. We report here that the enzyme contains one mol of a bound pterin cofactor/mol molybdenum, similar but not identical to the molybdopterin of milk xanthine oxidase. The two pterins, after oxidation with I2 at pH 2.5, showed identical fluorescence spectra and, after oxidation with permanganate at pH 13, yielded pterin 6-carboxylic acid. They differed, however, in their apparent molecular mass: the pterin of formylmethanofuran dehydrogenase was 400 Da larger than that of milk xanthine oxidase, a property also exhibited by the pterin cofactor of eubacterial molybdoenzymes. A homogeneous formylmethanofuran dehydrogenase preparation was used for these investigations. The enzyme, with a molecular mass of 220 kDa, contained 0.5-0.8 mol molybdenum, 0.6-0.9 mol pterin, 28 +/- 2 mol non-heme iron and 28 +/- 2 mol acid-labile sulfur/mol based on a protein determination with bicinchoninic acid. The specific activity was 175 mumol.min-1.mg-1 (kcat = 640 s-1) assayed with methylviologen (app. Km = 0.02 mM) as artificial electron acceptor. The apparent Km for formylmethanofuran was 0.02 mM.