It is now clear that oxygen-derived free radicals play an important part in several models of experimentally induced reperfusion injury. Although there are certainly multiple components to clinical ischemic and reperfusion injury, it appears likely that free-radical production may make a major contribution at certain stages in the progression of the injury. The primary source of superoxide in reperfused reoxygenated tissues appears to be the enzyme xanthine oxidase, released during ischemia by a calcium-triggered proteolytic attack on xanthine dehydrogenase. Reperfused tissues are protected in a variety of laboratory models by scavengers of superoxide radicals or hydroxyl radicals or by allopurinol or other inhibitors of xanthine oxidase. Dysfunction induced by free radicals may thus be a major component of ischemic diseases of the heart, bowel, liver, kidney, and brain.

Oxygen-derived free radicals in postischemic tissue injury.

Indirect evidence suggests accelerated degradation of endothelium-derived nitric oxide (ENDO) by superoxide anion (O2-) in hypercholesterolemic vessels (HV) To directly measure O2- production by normal vessels (NV) and HV, we used an assay for O2- based on the chemiluminescence (CL) of lucigenin (L) HV (1 mo cholesterol-fed rabbits) produced threefold more O2- than NV (147 +/- 020 nM/mg tissue/min, n = 7 vs 052 +/- 005 nmol/mg tissue/min, n = 8, P < 0001) Endothelial removal increased O2- production in NV (073 +/- 008, n = 6, P < 005), while decreasing it in HV (076 +/- 015, n = 5, P < 005) There was no difference between denuded HV and denuded NV Oxypurinol, a noncompetitive inhibitor of xanthine oxidase, normalized O2- production in HV, but had no effect in NV In separate isometric tension studies treatment with oxypurinol improved acetylcholine induced relaxations in HV, while having no effect on responses in normal vessels Oxypurinol did not alter relaxations to nitroprusside Thus, the endothelium is a source of O2- in hypercholesterolemia probably via xanthine oxidase activation Increased endothelial O2- production in HV may inactivate endothelium-derived nitric oxide and provide a source for other oxygen radicals, contributing to the early atherosclerotic process

/pdf/hypercholesterolemia-increases-endothelial-superoxide-anion-1rnwngjon2.pdf

Hypercholesterolemia increases endothelial superoxide anion production.

Toxic oxygen free radicals have been implicated as important pathologic mediators in many clinical disorders. We discuss the chemistry of oxygen radical production and the roles of iron an...

Oxygen Radicals and Human Disease

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.




Open in a separate window


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.

/pdf/the-mononuclear-molybdenum-enzymes-huemv59go8.pdf

The Mononuclear Molybdenum Enzymes

https://www.gastrojournal.org/article/0016-5085(81)90648-X/pdf

Superoxide radicals in feline intestinal ischemia

1. The ;xanthine oxidase' activity of rat liver supernatant, most of which behaves as an NAD(+)-dependent dehydrogenase (type D) can be rapidly converted into an oxidase (type O) by thiol reagents such as tetraethylthiuram disulphide, copper sulphate, 5,5'-dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide and p-hydroxymercuribenzoate. Treatment with copper sulphate, if prolonged, leads to almost complete inactivation of the enzyme. The effect of these reagents is prevented by dithioerythritol, and in all cases but that of N-ethylmaleimide is reversed by the same thiol. 2. Dithioerythritol prevents and reverses the conversion of xanthine oxidase from type D into type O brought about by storage of rat liver supernatant at -20 degrees C, preincubation under anaerobic conditions, treatment with carbon or with diethyl ether, and reverses, but does not prevent, the conversion obtained by preincubation of the whole liver homogenate. 3. Conversion of the enzyme from type D into type O is effected by preincubation of rat liver supernatant with the sedimentable fraction from rat liver but not from chick or pigeon liver. The xanthine dehydrogenase activity of chick liver supernatant is not changed into an oxidase by preincubation with the sedimentable fraction from rat liver. 4. The enzyme activity of rat liver supernatant is converted from type D into type O during purification of the enzyme: the purified enzyme can be reconverted into type D by dithioerythritol. 5. The enzyme appears as an oxidase in the supernatant of rat heart, intestine, spleen, pancreas, lung and kidney. The enzyme of all organs but intestine can be converted into a dehydrogenase by dithioerythritol.

/pdf/the-regulation-of-rat-liver-xanthine-oxidase-involvement-of-2qwd0uf4am.pdf

The regulation of rat liver xanthine oxidase. Involvement of thiol groups in the conversion of the enzyme activity from dehydrogenase (type D) into oxidase (type O) and purification of the enzyme.

/pdf/xanthine-oxidase-type-d-dehydrogenase-in-the-intestine-and-4vd96gekj0.pdf

Xanthine oxidase type D (dehydrogenase) in the intestine and other organs of the rat.

/pdf/regulation-of-xanthine-oxidase-in-rat-liver-modifications-of-4er4vtyavp.pdf

E D Corte

Papers

The regulation of rat liver xanthine oxidase. Involvement of thiol groups in the conversion of the enzyme activity from dehydrogenase (type D) into oxidase (type O) and purification of the enzyme.

Xanthine oxidase type D (dehydrogenase) in the intestine and other organs of the rat.

Regulation of xanthine oxidase in rat liver: modifications of the enzyme activity of rat liver supernatant on storage at 20 degrees.