Kinetic and structural studies of aldehyde oxidoreductase from Desulfovibrio gigas reveal a dithiolene-based chemistry for enzyme activation and inhibition by H(2)O(2)

Abstract

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 Desulfovibrio gigas (DgAOR) 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 DgAOR can be activated upon incubation with dithionite plus sulfide, a procedure similar to that used for activation of desulfo-XO. The fact that DgAOR does not need a sulfido ligand for catalysis indicates that the process leading to the activation of inactive DgAOR 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 DgAOR is activated. In contrast to XO, which is activated by resulfuration of the Mo site, DgAOR 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 DgAOR 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 η(2) fashion inhibiting the enzyme activity.

Figure 1. Schematic representation of the domains of bovine milk XO (PDB code: 1FIQ) (A) and DgAOR (B).

The Mo, FeS and FAD domains are depicted in green, red and yellow, respectively. The mechanisms of superoxide anion generation by XO as well as the proposal of peroxide generation and binding to the active site of DgAOR are represented. Figure was made using Qutemol .

Figure 2. Schematic representations of A) the active site of bovine milk XO and active-DgAOR and b) the structure of the pyranopterin cofactor present in both XO (R = H) and DgAOR (R =  cytidine monophosphate).

Figure 4. Crystallographic structures of A) inactive-DgAOR, B) activated-DgAOR, C) active-DgAOR crystals soaked with 30 mM sodium dithionite and 7 mM sodium sulfide after isopropanol removal (dit/S²⁻-soaked crystals), and D) active-DgAOR crystals soaked with 50 µM hydrogen peroxide (H₂O₂-soaked crystals).

Distances are in Å. Atoms color code: Mo in light teal, S in yellow, O in red, C in cyan. The 2mFo–DFc maps (blue mesh) are contoured at 1σ and the anomalous diffraction maps (orange mesh) are contoured at 3σ. The peroxide molecule was modeled in the three structures with occupancies of 1.0 for Ox1 and 0.5 for Ox2.

Figure 3. Normalized DgAOR activity vs. incubation time under aerobic (black circles) or anaerobic (empty circles) conditions.

Figure 5. Anomalous difference maps (orange mesh, contoured at 3σ) calculated from data collected at wavelength 2.06 Å for inactive-DgAOR crystal structure using A) 93 images (complete data set), B) the first 73 images, and C) the first 53 images.

Figure 6. Schematic representations of different pyranopterin forms of the molybdenum cofactor: the reduced tetrahydropyranopterin (Form I); 10,10a-dihydropyranopterin (Form II), a protonated form of the dihydro-pyranopterin possessing a S₇-thiolene/S₈-thione moiety (Form III), and a further one-electron oxidation of the dihydro-pyranopterin form is shown in Form IV.

Figure 7. EPR spectra of DMPO-hydroxyl radical (A) and DMPO-sulfite radical (B).

EPR parameters (g-values, A_N and A_H) were obtained through computer simulations.