Kinetic and Structural Studies of Aldehyde Oxidoreductase from <i>Desulfovibrio gigas</i> Reveal a Dithiolene-Based Chemistry for Enzyme Activation and Inhibition by H₂O₂

<div><p>Mononuclear Mo-containing enzymes of the xanthine oxidase (XO) family catalyze the oxidative hydroxylation of aldehydes and heterocyclic compounds. The molybdenum active site shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. The XO family member aldehyde oxidoreductase from <i>Desulfovibrio gigas</i> (<i>Dg</i>AOR) is an exception as presents in its catalytically competent form an equatorial oxo ligand instead of the sulfido ligand. Despite this structural difference, inactive samples of <i>Dg</i>AOR can be activated upon incubation with dithionite plus sulfide, a procedure similar to that used for activation of desulfo-XO. The fact that <i>Dg</i>AOR does not need a sulfido ligand for catalysis indicates that the process leading to the activation of inactive <i>Dg</i>AOR samples is different to that of desulfo-XO. We now report a combined kinetic and X-ray crystallographic study to unveil the enzyme modification responsible for the inactivation and the chemistry that occurs at the Mo site when <i>Dg</i>AOR is activated. In contrast to XO, which is activated by resulfuration of the Mo site, <i>Dg</i>AOR activation/inactivation is governed by the oxidation state of the dithiolene moiety of the pyranopterin cofactor, which demonstrates the non-innocent behavior of the pyranopterin in enzyme activity. We also showed that <i>Dg</i>AOR incubation with dithionite plus sulfide in the presence of dioxygen produces hydrogen peroxide not associated with the enzyme activation. The peroxide molecule coordinates to molybdenum in a η² fashion inhibiting the enzyme activity.</p>