Molybdenum enzymes structural studies

Of all of the molybdenum enzymes, mammalian xanthine oxidase/dehydrogenase has been the most studied (Figure 15). These studies, along with those of other members of this relatively large class of hydroxylases (Table la-c), suggest that all molybdenum enzymes that catalyze hydroxylation of C—H bonds contain a common structural motif. This motif is unique in high-valent molybdenum chem-... 

Mechanistic speculations about the molybdoenzymes must be considered to be in their infancy with the possible exception of those for xanthine oxidase. Although the detailed structural nature of the molybdenum site is unknown, there is sufficient information from biochemical and coordination chemistry studies to allow informed arguments to be drawn. Here we first discuss evidence for the nuclearity of the molybdenum site and then discuss both oxo-transfer and proton-electron transfer mechanisms for molybdenum enzymes. A final discussion considers the unique aspects of nitrogenase and the possible reasons for the use of molybdenum in enzymes. 

The most detailed spectroscopic and electronic structure studies of metallo-mono(dithiolenes) have focused on the nature of ligand-to-ligand charge transfer (LLCT) excitations in [M(diimine)(dithiolene)] complexes (112, 250-257, 262, 264, 295-301) and in monooxo molybdenum dithiolenes (19, 20, 22, 23) as models for pyranopterin molybdenum enzymes such as sulfite oxidase (SO). Since metallo-mono(dithiolenes) generally possess little or no symmetry, detailed spectrosopic and electronic structure studies of this class of metallo-dithiolenes have only recently begun to appear. The analysis of the spectroscopic data has been aided by the fact that the dithiolene-to-metal charge... 

Alcaligenes faecalis and five members of the p Proteobacteria are heterotrophic arsenite oxidizers, whereas Pseudomonas arsenitoxidans and NT-26 grew anaerobically through chemoautotrophic oxidation (Oremland and Stolz, 2005 Santini et al, 2000). However, six members of a Proteobacteria (Ben-5, NT-3, NT-4, NT-2, NT-26, and NT-25) and one member of y Proteobacteria (MLHE-1) were known chemohthoautotrophic arsenite oxidizers (Oremland et al, 2002). The best characterized and probably most studied of aU arsenite oxidizers is Alcaligenes faecalis, a heterotrophic arsenite oxidizer (Osborne and Enrlich, 1976). The arsenite oxidase from Alcaligenes faecalis has been purified and structurally characterized (Ellis et al, 2001). A similar enzyme has also been purified from the heterotrophic arsenite oxidizers Hydrogenophaga sp. strain NT-14 (Vanden Hoven and Santini, 2004) and the chemolithoautotrophic Rhizobium sp. strain NT-26 (Santini and Vanden Hoven, 2004), which indicate that the arsenite oxidase enzyme is also a member of the DMSO reductase family of molybdenum enzymes, similar to the respiratory arsenate reductases (Arr). The arsenite oxidase heterodimer comprises an 88 kDa catalytic subunit encoded by the aoxB gene that contains a [3Fe-4S] cluster and molybdenum bound to the pyranopterin cofactor and a 14 kDa subunit... 

The chapter consists of nine sections. Sections II through VII deal with the pterin-containing molybdenum enzymes. Biochemical and model studies of molybdopterin, Mo-co, and related species are described in Section II. In Section III, we briefly survey physical and spectroscopic techniques employed in the study of the enzymes, and consider their impact upon the current understanding of the coordination about the molybdenum atom in sulfite oxidase and xanthine oxidase. Model studies are described in Sections IV and V. Section IV concentrates on structural and spectroscopic models, whereas Section V considers aspects of the reactivity of model and enzyme systems. The xanthine oxidase cycle (Section VI) and facets of intramolecular electron transfer in molybdenum enzymes (Section VII) are then treated. Section VIII describes the pterin-containing tungsten enzymes and the evolving model chemistry thereof Future directions are addressed in Section IX. 

Molybdopterin itself is also extremely unstable when released from a protein and has never been structurally characterized in its native state (32, 33). Mass spectral and NMR studies of the difcarboxamido-methyl) derivative of the oxidized form of molybdopterin have provided convincing evidence that this derivative is a 6-substituted pterin that possesses structure 3 (34). A 6-substituted pterin moiety now appears to be a common feature of all of the molybdenum enzymes of Table I. There is still some question about the oxidation state of the pterin ring... 

In summary, a 6-substituted pterin was first identified as a structural component of the molybdenum cofactor from sulfite oxidase, xanthine oxidase and nitrate reductase in 1980 (24). Subsequent studies provided good evidence that these enzymes possessed the same unstable molyb-dopterin (1), and it seemed likely that 1 was a constituent of all of the enzymes of Table I. It now appears that there is a family of closely related 6-substituted pterins that may differ in the oxidation state of the pterin ring, the stereochemistry of the dihydropterin ring, the tautomeric form of the side chain, and the presence and nature of a dinucleotide in the side chain. In some ways the variations that are being discovered for the pterin units of molybdenum enzymes are beginning to parallel the known complexity of naturally occurring porphyrins, which may have several possible side chains, various isomers of such side chains, and a partially reduced porphyrin skeleton (46). 

In this section, an overview is of studies presented that revealed the presence of a pterin in molybdenum enzymes and eventually the structure of the pterin-dithiolene ligand of Moco. This background provides the context for pterin-inspired model work over the past three decades and highlights how the making of synthetic models for metal sites in enzymes follows acquisition of experimental data obtained from the metal site in the proteins. 

It required a decade of study to develop a clear understanding of the principles and outcome of pterin reactions to molybdenum. The results summarized here tell the story of non-innocent ligand behavior of pterin and pteridine ligands. During this period the first crystal structure of molybdenum enzymes emerged from which the dithiolene chelation of molyb-dopterin was proved. While protein structures unequivocally confirmed the dithiolene coordination to molybdenum, the alternative coordination mode described in this section underscores the electronic flexibility of the pterin system partnered with molybdenum. 

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