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

Abstract

Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a member of the xanthine oxidase (XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family 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. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V) EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerolinhibited enzyme, where the catalytically labile OH/OH 2 ligand is lost and both alcohols coordinate the Mo site in a η 2 fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system.

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

“…A similar phenomenon is observed in the XO inhibition by oxypurinol, where only the reduced sulfo-XO molecules are inhibited, in a process where the participation of the sulfhydryl group in the formation of the Mo-oxypurinol complex is not presently understood [40,49,50]. On the other hand, D. gigas AOR apparently does not need the sulfido group to catalyse the aldehyde oxidation [37] or the nitrite reduction. Understanding the role of the sulfido and oxo ligands during molybdenum reduction and oxidation is, thus, of prime importance in developing a deeper mechanistic insight into the XO-and AOR-mediated catalysis.…”

“…This enzyme possesses the same overall protein architecture, with two [2Fe-2S] centres similar to the XO ones, although it has no FAD [36]. AOR has the molybdenum atom in the same square-pyramidal geometry as XO, but its pterin cofactor is found esterificated with a cytidine monophosphate (pterin and cytosine dinucleotide) and it seems to have an =O group instead of the essential =S group of XO [37]. This enzyme catalyses the conversion of aldehydes to the respective carboxylates at the molybdenum site, and the Fe/S are involved in the intramolecular electron transfer to an unknown physiological oxidising substrate, probably the flavodoxin [38].…”

Molybdenum disulfide/graphene oxide nanocomposites show favorable lung targeting and enhanced drug loading/tumor-killing efficacy with improved biocompatibility

“…Chemotherapeutic agents that harbor ample groups including hydroxyl and amino, such as doxorubicin (DOX), are inclined to form coordinate linkage with Mo, 30,31 which renders MoS 2 as a desirable vehicle for drug delivery. In the meantime, given the poor dispersity of bulk MoS 2 and the inability to target specific tissue sites, more improvement therefore becomes very necessary for its biological applications, especially for targeted cancer nanotheranostics.…”

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

“…Electron paramagnetic resonance (EPR) studies performed on as-prepared DgAOR reduced with sodium dithionite showed that this pathway is able to transmit very weak exchange interactions [17,21,22,27]. Similar studies but performed on DgAOR samples treated with inhibitors such as glycerol (GOL) and ethylene glycol (EDO) showed that the J value depends on the nature of the inhibitor [31]. However, a detailed comparison of the structure of aspurified DgAOR with those reacted with GOL and EDO showed no significant changes in the nature and structural topology of the chemical path between Mo and FeS 1, which is in apparent contradiction with the fact that J depends on the structural characteristic of the chemical Fig.…”

“…Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a homodimeric mononuclear Mo-containing protein (*100 kDa per monomer) that belongs to the XO family, but does not contain an equatorial sulfido ligand at the Mo site [29][30][31]. Each of the monomers, which are catalytically independent [32], is organized into two major domains called Mo and FeS domains.…”