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Molybdenum (Mo) is an essential trace element used as cofactor in more than 60 enzymes from eukaryotes and prokariotes. All known Mo-containing enzymes may include two types structurally distingt cofactors: Mo-co and FeMoco. In most of Mo-containing proteins the Mo is complexed with a pterin moiety forming the molybdenum cofactor (Moco). FeMoco found only in Mo-containing nitrogenases which coordinates poly-metallic complex [MoFe7S9] [ 1, 2, 3, 4, 5 ]. All Mo-dependant enzymes are involved in biocycles of carbon, nitrogen, and sulfur of the Earth and are key components in several metabolic pathways. Molybdenum may have oxidation states in range of Mo2+ to Mo6+. In biological system molybdenum cycles between Mo4+, Mo5+ and Mo6+ oxidative states [ 2, 3, 5 ].

Under the normal atmospheric oxigen content, MoO42- and HMoO4-) are the most abundant chemical forms of Mo in oxygenated freshwater and soil [ 2, 3, 5 ]. The Recommended Dietary Allowance of molibdenum for adult men and women is 45-50 μg/day and 17-22 μg/day for chilrren. The average dietary intake of molybdenum by adult men and women is 109 and 76 μg/day, respectively. The Tolerable Upper Intake Level (UL) is 1.1-2 mg/day for adults and 0.3-0.6 mg/day (estimated based on impaired reproduction and growth in animals). The average molybdenum concentrations in whole blood is about 5 nmol/L. Plant foods is a major dietary source of Mo. Mo accumulation in plant varies depending upon the soil content in which they are grown. Legumes are major contributors of molybdenum in the diet, as well as grain products and nuts. Animal products, fruits, and many vegetables are generally low in molybdenum [ 6, 7 ].

Map of soil molybdenum content in the U.S. (red = high iron areas). Courtesy of U.S. Department of the Interior, U.S. Geological Survey, Mineral Resources.

Nitrogenase. Nitrogenase catalyzes the reduction of atmospheric N2 to NH3 as a key step of biological nitrogen fixation and key step in the global nitrogen cycle. Mo-dependent nitrogenases require the participation of two protein partners: nitrogenase reductase and the MoFe protein dinitrogenase [ 8, 9 ]. The catalized reaction is:

N2 + 8e- + 16ATP +8H+ → NH3 + N2 + 16ADP +16Pi;

[Fe4S3]-(bridging-S)3-[MoFe3S3] cofactor may function by two separate machanisms: (i) distal mechanism characterized by terminal nitride intermediate generation at the step of liberation of the first NH3 molecule, (ii) alternative mechanism [ 8, 9 ].

A schematic depiction of postulated N2 binding and reduction at an Fe site via limiting alternating (top) and distal (bottom) mechanisms. This picture emphasizes a possible hemi-labile role for the interstitial C-atom with respect to an Fe-N2 binding site [ 8 ].

Crystal structure of Nitrogenase from Azotobacter vinelandii. Iron-Sulfur-Molybdenum cluster is shown in blue color.

Molybdenum cofactor (Moco) containing enzymes. All known Moco-containing enzymes are oxidoreductases that transfer an oxo group or two electrons to or from the their substrates. There are more than 60 Moco-containing proteins have been characterize to date. Most of them are bacterial proteins. Molybdenum cofactor biosynthesis is an ancient, ubiquitous, and highly conserved pathway determining the biochemical activation of molybdenum. In different enzymes, molybdenum cofactor may have different structure derived from core Moco structure [ 10, 11, 12, 13 ].

Different structures of the molybdenum cofactor in E. coli [ 12 ].

(Moco) containing enzymes are divided into four families based on sequence similarity and spectroscopic properties and molybdenu cofactor structure: xanthine oxidases, sulfite oxidases, dimethylsulfoxide reductases, and aldehyde: ferredoxin oxidoreductases [ 11, 13, 14 ].

Xanthine oxidases family enzymes catalyze oxidative hydroxylation of a wide range of aldehydes and aromatic heterocycles. General reaction mechanism of these enzymes is base on reductive and oxidative half-reactions of the catalytic cycle. The major enzymes in this family include Xanthine oxidases which catalizes oxidation of hypoxanthine to xanthine and xanthine to uric acid, aldehyde oxidases which catalyzes the oxidation of wide range of aromatic and nonaromatic heterocycles and aldehydes, and bacterial aldehyde oxidoreductase. Xanthine oxidase family proteins are broadly distributed among eukaryotes, prokaryotes and archaea [ 2, 14, 15 ].

Proposed catalytic mechanism at the molybdenum site of xanthine oxidases [ 2 ].

Crystal structure of Bovine Xanthine Oxidase. Dioxomolybdenum (VI) ion is shown in blue color.

The sulfite oxidase family is represented by sulfite oxidases and assimilatory nitrate reductases. Sulfite oxidases catalyzes the oxidation of sulfite to sulfate as final step in the degradation of sulfur-containing amino acids. The assimilatory nitrate reductase catalyzes the reduction of nitrate to nitrite at the first step in the uptake and nitrate metabolism. Sulfite oxidases and nitrate reductase are typical for bacteria and eukaryotes [ 2, 5 ].

The dimethyl sulfoxide reductase (DMSOR) family represents bacterial and archaeal enzymes grouped based on substantial sequence homology. Dimethyl sulfoxide reductase catalyzes reduction of dimethyl sulfoxide (DMSO) to dimethyl sulfide (DMS), formate dehydrogenase catalyzes the oxidation of formate to bicarbonate, dissimilatory (or respiratory) nitrate reductase is responsable for the reduction of nitrate to nitrite, and trimethylamine-N-oxide (TMAO) reductases catalyzes the reduction of TMAO to trimethylamine. Arsenite oxidase and respiratory arsenate reductase are known to mediate the bioenergetic use of arsenics [ 2, 15, 16, 17, 18 ].

Proposed catalytic mechanism for DMSO reductase [ 16 ].

Crystal structure of DMSO reductase from Rhodobacter capsulatus. Molybdenum ion is shown in blue color, DMSO is shown in pink color.

Aldehyde: ferredoxin oxidoreductase is responsible for the interconversion of aldehydes and carboxylates in archaea [ 19, 20 ].

Molybdate uptake and regulation. Molybdenum is transported to the cell in the form of the molybdate (MoO42-). In E. coli molybdate may be transported using three different transport systems: (I) high affinity ModABC system, (II) low affinity CysPTWA sulfate-thiosulfate permease and (III) non-specific low-efficiency anion transport system that requires high molybdate concentrations, and which is involved in transports sulfate, selenate, and selenite [ 12, 21, 22, 23, 24, 25, 26 ]. The high-affinity molybdate transport system ModABC belongs to the ATP-binding cassette superfamily of transporters typical prokaryotes and eukaryotes. E. coli ABC transporter is formed by two integral membrane proteins ModB and two peripheral membrane proteins ModC that are ATP-binding proteins. Periplasmic protein ModA binds molybdate and delivers it to the transporter complex in the inner membrane [ 27, 28, 29 ]. modABC operon expression is negatively controlled by the ModE protein, which binds to the operator region of the modABC operon in its molybdate-bound form. ModE also enhances the transcription of molybdenum-dependent enzymes such as dimethylsulfoxide reductase reductase, nitrate reductase, formate hydrogen-lyase and molybdenum cofactor biosynthesis operon moaABCDE. [ 12, 30, 31 ]. Molybdate transport in eukaryotes is poorly understood and only one high-affinity molybdate transport system MOT1, which belongs to the sulfate transporter superfamily, has been characterized to date [ 32, 33, 34 ].

Schematics of Mo uptake and storage in prokaryotic and eukaryotic cells. Routes represented by dashed lines were only identified in prokaryotes, while routes represented by solid lines occurred in both current prokaryotes and eukaryotes. Green dots refer to reduced Mo species, and red dots refer to oxidized Mo (molybdate) [ 3 ].

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