Molybdenum enzymes sulfite oxidase

Sulfite Oxidase. This enzyme, isolated from bovine (26, 27) and chicken liver (28), catalyzes the oxidation of sulfite to sulfate. This is possibly a crucial function in animals as S032" (or S02, its gaseous precursor) is toxic while S042" is relatively innocuous. For example, one of the first signs of molybdenum deficiency in rats is a greatly increased susceptibility to S02 poisoning (28). In addition, a human child bom without sulfite oxidase activity did not survive for very long (29). 

Biochemical oxidation of sulfite to sulfate is mediated by a molybdoenzyme, sulfite oxidase. The enzyme activity has been reported in animals, plants, and certain bacteria. The enzyme contains Mo and heme Fe in the ratio 1 1. Molybdenum plays roles in both substrate binding and transfer of the O atom (Ochiai, 1987). 

Cohen et al. have shown that tungstate administered to pregnant rats inhibits the production of xanthine and sulfite oxidase (molybdenum-dependent enzymes) and high doses may be fatal to the fetus [28]. Accumulation of W is observed in maternal hypophise and ovaries and W added to the ovarian homogenates modifies the activity of adenylate cyclase. 

The enzymes that utilize molybdenum can be grouped into two broad categories (1) the nitrogenases, where Mo is part of a multinu-clear metal center, or (2) the mononuclear molybdenum enzymes, such as xanthine oxidase (XO), dimethyl sulfoxide (DMSO) reductase, formate dehydrogenase (FDH), and sulfite oxidase (SO). The last... 

Sulfite oxidase is a dimetallic enzyme that mediates the two-electron oxidation of sulfite by the one-electron reduction of cytochrome c. This reaction is physiologically essential as the terminal step in oxidative degradation of sulfur compounds. The enzyme contains a heme cofactor in the 10 kDa N-terminal domain and a molybdenum center in the 42 kDa C-terminal domain. The catalytic cycle is depicted in Fig. 9. 

Figure 17.2 The structure of the pterin cofactor (1) which is common to most molybdenum- and tungsten-containing enzymes and schematic active site structures for members of the xanthine oxidase (2,3), sulfite oxidase (4) and DMSO reductase (5-7) enzyme families. (From Enemark et al., 2004. Copyright (2004) American Chemical Society.)...

The reduction of DMSO catalyzed by molybdenum is an important step in the process of anaerobic respiration carried out by a number of bacteria (169). Much like sulfite oxidase, early MCD studies of DMSO reductase were complicated by the presence of heme iron (173). The discovery of two enzymes that do not include an iron center led to the measurement of MCD spectra of Rhodobacter sphaeroides DMSO reductase that could be assigned exclusively in terms of transitions of the Mo site (Fig. 10b) (174). The six major peaks are assigned as LMCT transitions from the three highest energy occupied orbitals to the two lowest unoccupied orbitals (174). 

The first hint of an essential role of molybdenum in metabolism came from the discovery that animals raised on a diet deficient in molybdenum had decreased liver xanthine oxidase activity. There is no evidence that xanthine oxidase is essential for all life, but a human genetic deficiency of sulfite oxidase or of its molybdopterin coenzyme can be lethal.646,646a,b The conversion of molybdate into the molybdopterin cofactor in E. coli depends upon at least five genes.677 In Drosophila the addition of the cyanolyzable sulfur (Eq. 16-64) is the final step in formation of xanthine dehydrogenase.678 It is of interest that sulfur (S°) can be transferred from rhodanese (see Eq. 24-45), or from a related mercaptopyruvate sulfurtransferase679 into the desulfo form of xanthine oxidase to generate an active enzyme.680... 

Nitrate reductase from Chlorella, an assimilatory enzyme, is a homotetramer of molecular weight 360 000 and contains one each of Mo, heme and FAD per subunit. The nitrate reductase from E. coli is a dissimilatory enzyme. EXAFS data are available on the molybdenum sites in both enzymes (Table 24).1050 The environment of the molybdenum in the assimilatory enzyme is similar to that found for sulfite oxidase, with at least two sulfur ligands near the molybdenum and a shuttle between monoxo and dioxo forms with redox change in the enzyme. This allows a similar mechanism to be put forward for the assimilatory nitrate reductase,1051 shown in equation (57), where an oxo group is transferred from nitrate to MoIV with production of nitrite and MoVI. 

The assimilatory nitrate reductase from Chlorella contains the molybdenum cofactor, as evidenced by the ability of the enzyme to donate the cofactor to the nitrate reductase of the mutant nit-1 of N. crassa. Reduction of the enzyme with NADH gives the Mov ESR signal, which is abolished on reoxidation with nitrate. Line shape and g values of the signal show a pH dependence similar to those observed previously for sulfite oxidase. The signal observed at pH 7.0 shows evidence for interaction with a single exchangeable proton.1053... 

Redox potentials of the molybdenum centers in several of the enzymes have been obtained by potentiometric titration (Table 3a). Although the substrate reaction chemistry requires the metal center to participate in net two-electron redox reactions, the simple electron-transfer reactions of the active sites occur in one-electron steps involving the MoVI/Mov and Mov/MoIV couples. Several of the molybdenum enzymes studied have MoVI/Mov and Mov/MoIV couples that differ by less than 40 mV. However, in sulfite oxidase the Movl/Mov (38 mV) and Mov/Molv (-239 mV) couples are separated by roughly 275 mV [88], In formate dehydrogenase (D. desulfuricans) the MoVI/Mov (-160 mV) and Mov/MoIV (-330 mV) couples are separated by 170 mV [89], Both the MoVI/Mov and... 

Protein sequence homology suggests that sulfite oxidase and assimilatory nitrate reductase are members of the same molybdenum enzyme subfamily [31]. Consistent with this classification, the cofactors of sulfite oxidase and assimilatory nitrate reductase differ significantly from those in dmso reductase, aldehyde oxido-reductase, xanthine oxidase (see Section IV.E.), and even respiratory nitrate reductase (Section IV.D). The EXAFS of both sulfite oxidase [132-136] and assimilatory nitrate reductase [131,137,138] and x-ray studies of sulfite oxidase (chicken liver) [116] confirm that the molybdenum center is coordinated by two sulfur atoms from a single MPT ligand and by the sulfur atom of a cysteine side chain. The Movl state is bis(oxido) coordinated (Figure 14). 

Mechanisms of action for the metal centers in acetylene hydratase, polysulfide reductase, and formate dehydrogenase have been briefly described in Sections VI.A and VLB. The discussion, in each case, was relatively straightforward insofar as the natures of these reactions lend themselves to simple mechanistic proposals. The mechanism by which the metal centers function in most of the other Mo and W enzymes is not as obvious. We elect to discuss mechanistic roles for the molybdenum centers in xanthine oxidase, sulfite oxidase, and dmso reductase. These enzymes are representative members of each large class of molybdenum enzymes, and the large body of literature on each enzyme makes detailed discussion possible. 

Molybdenum is a component of at least three enzymes aldehyde oxidase, xanthine dehydrogenase, and sulfite oxidase. The first two contain FAD, whereas the last is a heme protein similar to cytochrome c. Xanthine dehydrogenase can also act as an oxidase, that is, it can use 02 as an electron acceptor. Physiologically, however, it uses NAD+ as an electron acceptor when it converts hypoxanthine to xanthine and the latter to uric acid (see Chapter 10). Aldehyde and sulfite oxidases are true oxidases physiologically they both use 02 as an electron acceptor. Molybdenum in all three enzymes is associated with a pterinlike cofactor whose structure is shown in Figure 6.11. The Mo cofactor cannot be... 

The other molybdenum enzymes each contain duplicate prosthetic groups and paired subunits in addition to two molybdenum atoms. Many of the experiments performed for xanthine oxidase have also been carried out with aldehyde oxidase and sulfite oxidase, and there is no evidence for chemical Mo-Mo coupling in these enzymes. Thus, in oxidases, the evidence for mononuclear molybdenum sites appears strong, and in view of the duplicate subunits and composition found, it is reasonable to assume a similar situation in reductases as well. However, at present, insufficient information bars a full generalization. 

Current concepts of the chemical nature and role of molybdenum-containing enzymes are reviewed. Methods for molybdenum in enzymes, spectroscopic manifestations of the metal, and the characteristics of molybdenum-deficient enzymes are discussed, with particular attention to xanthine oxidase, sulfite oxidase, and nitrate reductase, in which Mo5 (and Mo3 in some cases) species are readily demonstrated. Nitrogenase is presumed to use molybdenum in a catalytic step, but no direct evidence for its participation in catalysis is yet available. 

The most detailed spectroscopic and electronic structure studies of metallo-mono(dithiolenes) have focused on the nature of ligand-to-ligand charge transfer (LLCT) excitations in [M(diimine)(dithiolene)] complexes (112, 250-257, 262, 264, 295-301) and in monooxo molybdenum dithiolenes (19, 20, 22, 23) as models for pyranopterin molybdenum enzymes such as sulfite oxidase (SO). Since metallo-mono(dithiolenes) generally possess little or no symmetry, detailed spectrosopic and electronic structure studies of this class of metallo-dithiolenes have only recently begun to appear. The analysis of the spectroscopic data has been aided by the fact that the dithiolene-to-metal charge... 

Figure 16. Consensus oxidized active-site structures of the xanthine oxidase (XO), sulfite oxidase (SO), and DMSO reductase (DMSOR), and aldehyde oxidoreductase (AOR) families of mononuclear molybdenum and tungsten enzymes and the structure of the common ppd cofactor (41, 42). The question mark in the AOR structure indicates uncertainty in the presence of a coordinated water molecule.

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