Superoxide Dismutase AN ENZYMIC FUNCTION FOR ERYTHROCUPREIN (HEMOCUPREIN)

Assaying for superoxide dismutase activity: Some large consequences of minor changes in conditions

Molybdenum is the only second-row transition metal required by most living organisms, and is nearly universally distributed in biology. Enzymes containing molybdenum in their active sites have long been recognized,1 and at present over 50 molybdenum-containing enzymes have been purified and biochemically characterized; a great many more gene products have been annotated as putative molybdenum-containing proteins on the basis of genomic and bioinformatic analysis.2 In certain cases, our understanding of the relationship between enzyme structure and function is such that we can speak with confidence as to the detailed nature of the reaction mechanism and, with the availability of high-resolution X-ray crystal structures, the specific means by which transition states are stabilized and reaction rate is accelerated within the friendly confines of the active site. At the same time, our understanding of the biosynthesis of the organic cofactor that accompanies molybdenum (variously called molybdopterin or pyranopterin), the manner in which molybdenum is incorporated into it, and then further modified as necessary prior to insertion into apoprotein has also (in at least some cases) become increasingly well understood.

It is now well-established that all molybdenum-containing enzymes other than nitrogenase (in which molybdenum is incorporated into a [MoFe7S9] cluster of the active site) fall into three large and mutually exclusive families, as exemplified by the enzymes xanthine oxidase, sulfite oxidase, and DMSO reductase; these enzymes represent the focus of the present account.3 The structures of the three canonical molybdenum centers in their oxidized Mo(VI) states are shown in Figure 1, along with that for the pyranopterin cofactor. The active sites of members of the xanthine oxidase family have an LMoVIOS-(OH) structure with a square-pyramidal coordination geometry. The apical ligand is a Mo=O ligand, and the equatorial plane has two sulfurs from the enedithiolate side chain of the pyranopterin cofactor, a catalytically labile Mo–OH group, and most frequently a Mo=S. Nonfunctional forms of these enzymes exist in which the equatorial Mo=S is replaced with a second Mo=O; in at least one member of the family the Mo=S is replaced by a Mo=Se, and in others it is replaced by a more complex –S–Cu–S–Cys to give a binuclear center. Members of the sulfite oxidase family have a related LMoVIO2(S–Cys) active site, again square-pyramidal with an apical Mo=O and a bidentate enedithiolate Ligand (L) in the equatorial plane but with a second equatorial Mo=O (rather than Mo–OH) and a cysteine ligand contributed by the protein (rather than a Mo=S) completing the molybdenum coordination sphere. The final family is the most diverse structurally, although all members possess two (rather than just one) equiv of the pyranopterin cofactor and have an L2MoVIY(X) trigonal prismatic coordination geometry. DMSO reductase itself has a catalytically labile Mo=O as Y and a serinate ligand as X completing the metal coordination sphere of oxidized enzyme. Other family members have cysteine (the bacterial Nap periplasmic nitrate reductases), selenocysteine (formate dehydrogenase H), –OH (arsenite oxidase), or aspartate (the NarGHI dissimilatory nitrate reductases) in place of the serine. Some enzymes have S or even Se in place of the Mo=O group. Members of the DMSO reductase family exhibit a general structural homology to members of the aldehyde:ferredoxin oxidoreductase family of tungsten-containing enzymes;4 indeed, the first pyranopterin-containing enzyme to be crystallographically characterized was the tungsten-containing aldehyde:ferredoxin oxidoreductase from Pyrococcus furiosus,5 a fact accounting for why many workers in the field prefer “pyranopterin” (or, perhaps waggishly, “tungstopterin”) to “molybdopterin”. The term pyranopterin will generally be used in the present account.




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Figure 1


Active site structures for the three families of mononuclear molybdenum enzymes. The structures shown are, from left to right, for xanthine oxidase, sulfite oxidase, and DMSO reductase. The structure of the pyranopterin cofactor common to all of these enzymes (as well as the tungsten-containing enzymes) is given at the bottom.

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The Mononuclear Molybdenum Enzymes

Quantitative aspects of the production of superoxide anion radical by milk xanthine oxidase

There is substantial evidence that oxidative stress participates in the pathophysiology of cardiovascular disease. Biochemical, molecular and pharmacological studies further implicate xanthine oxidoreductase (XOR) as a source of reactive oxygen species in the cardiovascular system. XOR is a member of the molybdoenzyme family and is best known for its catalytic role in purine degradation, metabolizing hypoxanthine and xanthine to uric acid with concomitant generation of superoxide. Gene expression of XOR is regulated by oxygen tension, cytokines and glucocorticoids. XOR requires molybdopterin, iron-sulphur centres, and FAD as cofactors and has two interconvertible forms, xanthine oxidase and xanthine dehydrogenase, which transfer electrons from xanthine to oxygen and NAD(+), respectively, yielding superoxide, hydrogen peroxide and NADH. Additionally, XOR can generate superoxide via NADH oxidase activity and can produce nitric oxide via nitrate and nitrite reductase activities. While a role for XOR beyond purine metabolism was first suggested in ischaemia-reperfusion injury, there is growing awareness that it also participates in endothelial dysfunction, hypertension and heart failure. Importantly, the XOR inhibitors allopurinol and oxypurinol attenuate dysfunction caused by XOR in these disease states. Attention to the broader range of XOR bioactivity in the cardiovascular system has prompted initiation of several randomised clinical outcome trials, particularly for congestive heart failure. Here we review XOR gene structure and regulation, protein structure, enzymology, tissue distribution and pathophysiological role in cardiovascular disease with an emphasis on heart failure.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.2003.055913

Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications

https://www.jbc.org/content/245/11/2837.full.pdf

Hirochika Komai

Papers

On the Mechanism of Inactivation of Xanthine Oxidase by Allopurinol and Other Pyrazolo[3,4-d]pyrimidines

Studies on Milk Xanthine Oxidase: Some spectral and kinetic properties

The Preparation and Properties of Deflavo Xanthine Oxidase