The crystal structure of xanthine oxidoreductase during catalysis: Implications for reaction mechanism and enzyme inhibition

Abstract

Molybdenum is widely distributed in biology and is usually found as a mononuclear metal center in the active sites of many enzymes catalyzing oxygen atom transfer. The molybdenum hydroxylases are distinct from other biological systems catalyzing hydroxylation reactions in that the oxygen atom incorporated into the product is derived from water rather than molecular oxygen. Here, we present the crystal structure of the key intermediate in the hydroxylation reaction of xanthine oxidoreductase with a slow substrate, in which the carbon-oxygen bond of the product is formed, yet the product remains complexed to the molybdenum. This intermediate displays a stable broad charge-transfer band at Ϸ640 nm. The crystal structure of the complex indicates that the catalytically labile MoOOH oxygen has formed a bond with a carbon atom of the substrate. In addition, the MoAS group of the oxidized enzyme has become protonated to afford MoOSH on reduction of the molybdenum center. In contrast to previous assignments, we find this last ligand at an equatorial position in the square-pyramidal metal coordination sphere, not the apical position. A water molecule usually seen in the active site of the enzyme is absent in the present structure, which probably accounts for the stability of this intermediate toward ligand displacement by hydroxide.

Nitrite reduction by xanthine oxidase family enzymes: a new class of nitrite reductases

“…6a), at the molybdenum centre (Scheme 1b, i ? iii) [12,15,16]. The electrons thus introduced into the molybdenum are subsequently transferred to the Fe/S and FAD.…”

“…The hydroxylation catalysis [15,16] is initiated with the activation of the molybdenum-labile -OH/OH 2 group by a neighbouring unprotonated glutamate residue (Glu-1261 in the bovine enzyme) to form Mo 6? -O -(base-assisted catalysis) (Scheme 1b, i).…”

“…The participation of the XO and AOR redox centres in nitrite reduction was assessed by EPR spectroscopy, in the presence of typical reducing substrates (aldehydes, xanthine and NADH), dithionite and site-specific inhibitors (allopurinol and ethylene glycol) and with samples of desulfo-XO (an enzymatic form in which the essential Mo=S was replaced by a Mo=O group). The kinetic and spectroscopic results were integrated with the available structural and mechanistic data [7,12,15,16] to suggest a molecular mechanism of molybdenum-dependent nitrite reduction.…”

Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo−C Interaction in a Biological System

“…(d) Base-assisted nucleophilic attack mechanism. 6,13 (e) Electrophilic attack mechanism as suggested by Howes et al on the basis of ENDOR studies. 14 lyzable sulfido ligand can be reinserted with regain of the enzyme activity.…”

“…(1) Hille ligand, 6,[11][12][13] where the nearby glutamic acid residue acts as base for the proton of the OH x ligand, enhancing its nucleophilic character. Structures of the most relevant intermediates of this proposal are given in Figure 1d.…”