Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase

Abstract

Two arsenite-inhibited forms of each of the aldehyde oxidoreductases from Desulfovibrio gigas and Desulfovibrio desulfuricans have been studied by X-ray crystallography and electron paramagnetic resonance (EPR) spectroscopy. The molybdenum site of these enzymes 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. Arsenite addition to active as-prepared enzyme or to a reduced desulfo form yields two different species called A and B, respectively, which show different Mo(V) EPR signals. Both EPR signals show strong hyperfine and quadrupolar couplings with an arsenic nucleus, which suggests that arsenic interacts with molybdenum through an equatorial ligand. X-ray data of single crystals prepared from EPR-active samples show in both inhibited forms that the arsenic atom interacts with the molybdenum ion through an oxygen atom at the catalytic labile site and that the sulfido ligand is no longer present. EPR and X-ray data indicate that the main difference between both species is an equatorial ligand to molybdenum which was determined to be an oxo ligand in species A and a hydroxyl/water ligand in species B. The conclusion that the sulfido ligand is not essential to determine the EPR properties in both MoAs complexes is achieved through EPR measurements on a substantial number of randomly oriented chemically reduced crystals immediately followed by X-ray studies on one of those crystals. EPR saturation studies show that the electron transfer pathway, which is essential for catalysis, is not modified upon inhibition.

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

“…X-ray studies show that both alcohol molecules are stabilized in their respective positions by a hydrogen bond with the O atom from Glu869 (see Figure 3). A similar feature was also observed for the arsenite moiety in the structure of arsenite-inhibited DgAOR 7,8 and for substrates in closely related proteins. 5,13 Additional proof of the stability of the Mo-alcohol adducts is given by the fact that inhibitor molecules cannot be easily removed once they entered into the active site pocket, as indicated by the extensive washing necessary for the enzyme to regain activity.…”

“…This indicates that either oxidized Mo(VI) or reduced Mo(IV) forms can react with the alcohol molecules, similar to what occurs for arsenite inhibition. 8 ENDOR studies of normal and deuterated EDOtreated XO detected a hyperfine coupling of 3.6 MHz (∼1.2 G) that was assigned to the protons of the CH 2 groups, confirming that the ethylene glycol is coordinated to Mo in the EPR-active species. 52 On the basis of this result, it was suggested that the Mo(V) ion is coordinated through both oxygen atoms of the alcohol moiety.…”

“…7,8,23,27 These inhibited forms are important to understand changes in the coordination of the active site and the integrity of the electron transfer chain upon inhibition. In previous structural and EPR studies of arsenite-inhibited DgAOR, a correlation between structural and EPR properties of the Mo center was established.…”

“…In previous structural and EPR studies of arsenite-inhibited DgAOR, a correlation between structural and EPR properties of the Mo center was established. 7,8 The major findings of these studies were to elucidate the interaction between the inhibitory arsenite ion and the molybdenum atom ( Figure 1c) and to establish that the sulfido ligand is not essential to determine the EPR properties of the arsenic-bound molybdenum center. Particularly, the latter raised some doubts about the presence of the sulfido ligand in active DgAOR.…”

Isotropic exchange interaction between Mo and the proximal FeS center in the xanthine oxidase family member aldehyde oxidoreductase from Desulfovibrio gigas on native and polyalcohol inhibited samples: an EPR and QM/MM study

“…For biological systems, the evaluation of J is not only relevant to understand the electronic properties of the redox centers, but also to learn on structural and functional aspects of the systems that complement information obtained from conventional structural and kinetic techniques [18][19][20]. Particularly, the analysis of the magnetic interaction between centers can be advantageously used to determine intercenter distances, to assign the EPR active centers with those of the structure, and to study the integrity of the electron transfer pathways in distinct protein conditions [21][22][23][24][25][26]. Additional interest in the evaluation of exchange interactions is that the rate of electron transfer through a chemical path linking two paramagnetic centers is proportional to |J| under conditions of maximal rate, which means that the value of |J| can be advantageously used to predict the ability of the chemical path as electron transfer conduit [23].…”