Arsenite oxidoreductase

The arsenite oxidoreductase from A. faecalis (NCIB 8687) has been purified, characterized (15), and the structure recently determined by x-ray crystallography... 

Immunological precipitation of arsenite oxidoreductase indicates that the enzyme is induced by arsenite but not by arsenate (L. A. Kimpler and G. L. Anderson, unpublished data), suggesting a separate mechanism for cell survival in the presence of arsenate. 

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.

Figure 3 Visible absorption spectra of oxidized and reduced arsenite oxidoreductase. (A) Visible spectrum of oxidized and reduced arsenite oxidoreductase in 50 mM MBS, pH 6.0. (B) Difference spectrum of oxidized minus reduced arsenite oxidoreductase. (From Ref. 15.)...

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.

The x-ray crystal structure of arsenite oxidoreductase from A. faecalis has recently been determined (16). The protein was found to be a heterodimer in each... 

Figure 6 Stereoview ribbon diagram of arsenite oxidoreductase from Alcaligenes faecalis. The enzyme is comprised of a large subunit consisting of four domains designated by roman numerals and a small or Rieske subunit. The metal sites in the enzyme are shown as atomic spheres. The molecule is oriented to look down the solvent access channel at the Mo active site.

Figure 7 Proposed reaction mechanism of arsenite oxidation in arsenite oxidoreductase.

The small subunit of arsenite oxidoreductase, consisting of 134 amino acid residues, can be divided into two domains, each consisting principally of P-sheet (see Fig. 6). The overall fold is similar to the Rieske-containing subunits of cytochrome ( e/(49) and bci (50,51), and the Rieske-containing domain of phthalate-... 

The steady state kinetics of arsenite oxidoreductase from A. faecalis indicate a so-called double displacement (or ping-pong ) mechanism (15) in which the enzyme cycles between oxidized and reduced forms in its reaction with arsenite and azurin (or cytochrome c). This overall kinetic scheme is common in redox-active proteins. Arsenite must bind, the oxygen atom transfer chemistry take place, and arsenate dissociate before the subsequent reaction of a second molecule of substrate. Since arsenate is not an inhibitor of arsenite oxidoreductase (43), product dissociation must be effectively irreversible. The turnover number (kcai) of 27 sec and for arsenite of 8 pM are reasonable parameters for the detoxification of arsenite, especially since A. faecalis is able to survive in at least 80 mM (1%) sodium arsenite. The considerable catalytic power of the enzyme is reflected by the kinetic parameter k JK of 3.4 X 10 M sec , which is fairly close to the diffusion-controlled maximum of 10 -10 M sec for proteins in... 

The activity of arsenite oxidoreductase from A. faecalis is affected by essential histidines (43). Approximately three histidine residues in the oxidized enzyme are readily accessible to chemical modification by diethylpyrocarbonate, and at least one of these modulates the activity of the oxidized enzyme. However, if arsenite oxidoreductase is first reduced by either dithionite (a low potential generic reductant) or by arsenite, approximately three histidines can be modified, without affecting arsenite oxidoreductase activity. The reductive half reaction of arsenite oxidoreductase may therefore be dependent on histidine residue(s) either for the process of electron transfer or for the correct conformation of the oxidized protein. As indicated above. His 195 and His423 form part of the binding site and one of these may be the residue whose modification in oxidized enzyme results in loss of activity. 

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