Structural and Electron Paramagnetic Resonance (EPR) Studies of Mononuclear Molybdenum Enzymes from Sulfate-Reducing Bacteria

Abstract

Molybdenum and tungsten are found in biological systems in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen-atom-transfer reactions. The metal atom (Mo or W) is coordinated to one or two pyranopterin molecules and to a variable number of ligands such as oxygen (oxo, hydroxo, water, serine, aspartic acid), sulfur (cysteines), and selenium (selenocysteines) atoms. In addition, these proteins contain redox cofactors such as iron-sulfur clusters and heme groups. All of these metal cofactors are along an electron-transfer pathway that mediates the electron exchange between substrate and an external electron acceptor (for oxidative reactions) or donor (for reductive reactions). We describe in this Account a combination of structural and electronic paramagnetic resonance studies that were used to reveal distinct aspects of these enzymes. Mononuclear Molybdenum Enzymes: Classification and General PropertiesMolybdenum and tungsten are found in biological systems in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen-atom-transfer reactions. 1,2 Mononuclear molybdenum-containing enzymes are ubiquitous and participate in several biological processes occurring in nature, such as denitrification, the greenhouse effect, and pollution of the water soil. 3,4,5 Mononuclear molybdenum enzymes have been divided into three groups called the xanthine oxidase (XO), dimethyl sulfoxide reductase (DMSOR), and sulfite oxidase (SO) families. 1,6 These three families include not only the enzymes that give the name to the different groups but also diverse enzymes, such as aldehyde oxidoreductases, nitrate reductases, and formate dehydrogenases, among others. The active site of these enzymes (Figure 1a) includes the metal atom coordinated to one or two pyranopterin molecules and to a variable number of ligands such as oxygen (oxo, hydroxo, water, serine, aspartic acid), sulfur (cysteines), and selenium (selenocysteines) atoms. The pyranopterin molecule is an organic ligand that can be either in the monophosphate form or have a nucleotide molecule attached by a pyrophosphate link (Figure 1b). 7 In addition, these proteins may also have other redox cofactors such as iron-sulfur (FeS) centers, hemes, and flavin groups, which are involved in intra-and intermolecular electron-transfer processes. A tungstencontaining aldehyde oxidoreductase from Pyrococcus furiosus presents a similar active-site structure and has been classified in a different family. 2 However, other tungsten enzymes such as the formate dehydrogenase from Desulfovibrio gigas belong to the DMSOR family, being closely related to the corresponding molybdenum enzymes. 8 With a few exceptions, these enzymes catalyze the transfer of an oxygen atom from water to the product (or vice versa) in reactions that imply a net exchange of two electrons between the enzyme and substrate and in which the metal ion cycles between the redox states IV and VI. Figure 2 shows two representative exam...

The mechanism of formate oxidation by metal-dependent formate dehydrogenases

“…This type of behavior was also proposed for the catalytic mechanism of the periplasmic nitrate reductase, where the nitrate anion binds a similar negatively charged active site [37]. These results suggest that one of the roles of the pyranopterinic cofactors in these enzymes might be, in addition to providing an electron transfer pathway [9,10], to cushion the electrostatic repulsion produced by the negative charge of formate, lowering the activation energy barrier for the formation of the ES complex.…”

“…In the second case (not shown), it was assumed that after the transfer of the proton of the formate to the SeCys residue, two electrons would be immediately transferred to the redox centers included in the electron transfer pathway of the protein [9,10]. This implies the two-electron oxidation of the active site, for which it was necessary to manually adjust the charge of the model to ?1.…”

“…The crystal structures of three of these enzymes have been reported. The molybdenumcontaining enzymes are Fdh-H and the membrane-bound Fdh-N from Escherichia coli K12 [4][5][6][7][8][9][10]. The tungstencontaining enzyme is the periplasmic Fdh from the sulfatereducing bacterium Desulfovibrio gigas [9][10][11][12].…”

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

“…Molybdenum is present in a wide variety of enzymes, in both prokaryotes and eukaryotes, where it performs catalytic roles in important redox reactions of the carbon, nitrogen and sulfur cycles [1][2][3]. Additionally, heterometallic clusters of molybdenum are also found in other proteins whose physiological function is not yet known [4,5].…”

“…With the exception of the unique iron-molybdenum cofactor of the nitrogenase, molybdenum is found in enzyme catalytic sites coordinated by the cis-dithiolene group of one or two pterin cofactor 1 molecules (Scheme 1a) [1][2][3]. The coordination sphere of the molybdenum atom is completed with oxygen, sulfur or selenium atoms in a diversity of arrangements (see below).…”