Molybdenum arsenites

Molybdenum Arsenites have not been prepared, but complex molybdoarsenites similar to the tungstoarsenites are known. 

Johnson and Pilson [229] have described a spectrophotometric molybdenum blue method for the determination of phosphate, arsenate, and arsenite in estuary water and sea water. A reducing reagent is used to lower the oxidation state of any arsenic present to +3, which eliminates any absorbance caused by molybdoarsenate, since arsenite will not form the molybdenum complex. This results in an absorbance value for phosphate only. 

Some compounds of this type may have a high affinity for proteins that is not due to their binding to two thiol groups (35). In particular, arsenite also reacts with the molybdenum-pterin cofactor of many enzymes (35a-d). This usually inhibits the enzyme, but in particular cases (35e) the arsenite may be oxidized indeed the enzyme arsenite oxidase contains such a center (35f). 

Reducing agents (sulphides, thiosulphates, etc.) interfere since they yield molybdenum blue hexacyanoferrate(II) ions give a red colouration. Arsenates (warming is usually required), arsenites, chromates, oxalates, tartrates, and silicates give a similar reaction with some variation in the colour of the precipitate. All should be removed before applying the test. 

Coughlan, M. P., Rajagopalan, K. V., and Handler, P., 1969, The role of molybdenum in xanthine oxidase and related enzymes. Reactivity with cyanide, arsenite, and methanol, J. Biol. Chem. 244 2658112663. 

Alcaligenes faecalis and five members of the p Proteobacteria are heterotrophic arsenite oxidizers, whereas Pseudomonas arsenitoxidans and NT-26 grew anaerobically through chemoautotrophic oxidation (Oremland and Stolz, 2005 Santini et al, 2000). However, six members of a Proteobacteria (Ben-5, NT-3, NT-4, NT-2, NT-26, and NT-25) and one member of y Proteobacteria (MLHE-1) were known chemohthoautotrophic arsenite oxidizers (Oremland et al, 2002). The best characterized and probably most studied of aU arsenite oxidizers is Alcaligenes faecalis, a heterotrophic arsenite oxidizer (Osborne and Enrlich, 1976). The arsenite oxidase from Alcaligenes faecalis has been purified and structurally characterized (Ellis et al, 2001). A similar enzyme has also been purified from the heterotrophic arsenite oxidizers Hydrogenophaga sp. strain NT-14 (Vanden Hoven and Santini, 2004) and the chemolithoautotrophic Rhizobium sp. strain NT-26 (Santini and Vanden Hoven, 2004), which indicate that the arsenite oxidase enzyme is also a member of the DMSO reductase family of molybdenum enzymes, similar to the respiratory arsenate reductases (Arr). The arsenite oxidase heterodimer comprises an 88 kDa catalytic subunit encoded by the aoxB gene that contains a [3Fe-4S] cluster and molybdenum bound to the pyranopterin cofactor and a 14 kDa subunit... 

Santini, J.M., Vanden Hoven, R.N. (2004). Molybdenum-containing arsenite oxidase of the chemolithoautotrophic arsenite oxidizer NT-26. J. Bacteriol. 186 1614-19. 

The carbonates, sulphates, and borates are decomposed. The sulphides of the alkalies and alkaline earths are decomposed while the sulphides of arsenic, antimony, molybdenum, zinc, cadmium, tin, iron, lead, copper, mercury, and palladium are not attacked. Cobalt sulphate is not attacked, while the sulphates of the alkalies and alkaline earths are attacked and dissolved. Alkali tungstates, ammonium arsenite and arsenate, copper arsenite, ammonium magnesium arsenate, ammonium molybdate and vanadate, potassium cyanide and ferrocyanide are decomposed. Paraffin is not attacked shellac, gum arabic, gum tragacanth, copal, etc., are decomposed. Celluloid is slowly attacked. Silk paper, gun cotton, gelatin, parchment are dissolved. M. Meslans 22 has studied the esterification of alcohol by hydrofluoric acid. 

In this section are described the important chemical features of those substrates which are oxidized by the molybdenum hydroxylases. Although these enzymes, particularly aldehyde oxidase, also catalyse numerous reductive reactions under anaerobic conditions in vitro, it has not yet been established whether they occur under physiological conditions and there are as yet insufficient examples of any one reduction reaction to permit any conclusions regarding the structure of substrates. Thus, such reactions will not be discussed here (see [11] and references therein). Properties of those inhibitors which bind at the Mo centre and are also substrate analogues will also be included. However, the interaction of inhibitors such as cyanide and arsenite with the molybdenum hydroxylases and the mechanism of action of the specific xanthine oxidase inhibitor, allo-purinol, have been comprehensively described elsewhere [8, 12, 14, 157]. 

Fig. 15.8b has been used for the speciation or arsenic (as arsenite and arsenate) by use of the Molybdenum Blue reaction after complexation of As(V) with Mo(VI). A KIOs solution is used to oxidize AsOr in this case. The calibration garphs are linear In the range lO MO M for both ions and mixtures in ratios up to 20 1 can be readily resolved [38]. 

Andeeson GL, Williams J and Hille R (1992) The purification and characterization of arsenite oxidase from Alcaligenesfaecalis, a molybdenum-containing hydroxylase. J Biol Chem 267 23674-23682. 

Arsenite oxidase of Alicaligenes faecalis is comprised of a large subunit that contains a molybdenum center that is very similar to that of the nitrate reductase, a Fe3S4 cluster, plus a smaller unit containing a Reiske-type Fc2S2 cluster. 

The arsenite oxidase is a molybdenum-containing hydroxylase (63,64) with two [Fe-S] centers (see Fig. 8). It is found in the periplasmic space between the inner and outer membranes of Alcaligenes, coupled via the small blue copper protein azurin to cytochrome c (63). Arsenite oxidase consists of a large subunit... 

The inducible arsenite oxidase from the Eubacterium Alcaligenes faecalis (NCIB 8687) has been purified and characterized (22-24). Anderson et al. (24) isolated the enzyme from a sonicate of washed, lysozyme-treated cells that had been harvested in their late exponential growth phase. The sonicate was fractionated by gel filtration through DEAE-sepharose and active fractions concentrated by ultrafiltration. The purified enzyme was found to be monomeric with a molecular mass of 85 kDa. It consisted of two polypeptide chains in an approximate ratio of 70 30. The enzyme stmcture included one molybdenum, five or six iron atoms, and sulfide. Purification of the oxidase also led to recovery of azurin, a blue protein, which was rapidly reduced by arsenite in the presence of catalytic amounts of Aro, and a red protein. The red protein was a c-type cytochrome, which was reduced by arsenite in the presence of catalytic amounts of Aro and azurin. No reduction of the cytochrome occurred in the absence of Aro, but it did occur in the absence of azurin. Denaturation of Aro led to the release of a pterin cofactor characteristic of molybdenum hydroxylases. In intact cells of A. faecalis, the enzyme resides on the outer surface of the inner (plasma) membrane. The cytochrome and azurin may be part of an electron transfer pathway in the periplasm. 

JW Williams, SJ Rinderle, JA Schrier, LJ Alvey, K Tseng. Arsenite oxidase A molybdenum-containing iron-sulfur protein (abstr 1050). Fed Proc 45 1660, 1986. GL Anderson, J Williams, R Hille. The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase. J Biol Chem 267 23674-23682, 1992. 

Figure 4 Sequence alignment of the N-termini of AroA and molybdenum-containing proteins. The sequences belong to the arsenite oxidase of NT-26 (NT-26 AroA), the formate dehydrogenase of Wolinella succinogenes (W.s. FdhA), and the nitrate-inducible formate dehydrogenase of Escherichia coli (E.c. FdnG). Boxed amino acids show identity to NT-26 AroA.

Since detoxification of arsenite occurs via oxidation to arsenate, understanding the mechanism of arsenite oxidoreductase has centered on the three redox-active centers found in the enzyme. These are a molybdenum center, a [3Fe S] cluster. 

Figure 2 Iron-sulfur clusters and molybdenum cofactor of arsenite oxidoreductase. The moiybdopterin cofactor refers to the organic moiety, excluding the molybdenum atom. R = guanine mononucleotide in arsenite oxidoreductase.

Since it is well established that electron transfer into or out of other molybdenum-containing proteins occurs at the molybdenum center (32-36), this is the most likely site for the binding of arsenite in the case of arsenite oxidoreductase. Eur-thermore, arsenite is a potent inhibitor of some MPT-containing enzymes (37,38), and for xanthine oxidase has been shown unequivocally to bind within the coordi-... 

Figure 5 Structure of the molybdenum center in xanthine oxidase. The sulfido ligand to molybdenum is the site at which arsenite binds in xanthine oxidase. This group is absent in arsenite oxidoreductase.

A (39). In addition, the Mo(V) EPR spectrum of arsenite-inhibited xanthine oxidase clearly shows hyperfine structure from the As nucleus (40,41). In binding at the molybdenum center, arsenite blocks the active site and inhibits electron transfer activity in xanthine oxidase via the effect it has on the relative reduction potentials of the redox-active centers (41). 

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