Cytochrome reaction with xanthine oxidase

The role of these interesting plasma membrane-dependent, vanadate-stimulated NAD(P)H oxidation reactions in cellular metabolism remains to be elucidated, although multiple interactions with cellular metabolism and components are possible including interactions with xanthine oxidase and lipid peroxidation [24], Decavanadate has been shown to enhance cytochrome c reduction [31], and cytochrome c release from mitochondria is associated with initiation of apoptosis. Perhaps the reduced cytochrome c is more readily released from the mitochondria. With increasing emphasis on the redox properties of vanadium being important in its pharmacological effects, it is quite possible that these reactions, either protein dependent or not, may play a role in therapeutic actions of vanadium. 

The reduced form of xanthine oxidase is oxidized by molecular oxygen or by methylene blue. It will also react with cytochrome c at a rate which is about one-half the rate with oxygen. However, the reduction of cytochrome c is dependent on the presence of oxygen, and very little reaction occurs under anaerobic conditions. This requirement of oxygen for the reduction of cytochrome c by xanthine oxidase has been explained in terms of the formation of a free radical in the oxidation of the leucoflavoprotein. Oxygen would be necessary to form the free radical from the leucoform, but the free radical could react with either oxygen or cytochrome c.f... 

Superoxide is formed (reaction 1) in the red blood cell by the auto-oxidation of hemoglobin to methemo-globin (approximately 3% of hemoglobin in human red blood cells has been calculated to auto-oxidize per day) in other tissues, it is formed by the action of enzymes such as cytochrome P450 reductase and xanthine oxidase. When stimulated by contact with bacteria, neutrophils exhibit a respiratory burst (see below) and produce superoxide in a reaction catalyzed by NADPH oxidase (reaction 2). Superoxide spontaneously dismu-tates to form H2O2 and O2 however, the rate of this same reaction is speeded up tremendously by the action of the enzyme superoxide dismutase (reaction 3). Hydrogen peroxide is subject to a number of fates. The enzyme catalase, present in many types of cells, converts... 

Possible errors due to the competition of cytochrome c reduction with the reversible reduction of quinones by superoxide are frequently neglected. For example, it has been found that quinones (Q), benzoquinone (BQ), and menadione (MD) enhanced the SOD-inhibitable cytochrome c reduction by xanthine oxidase [6]. This seems to be a mystery because only menadione may enhance superoxide production by redox cycling ( °p)"]/ [MD] =-0.20 V against ,0[02 ]/[02] 0.16 V) via Reactions (3) and (4), whereas for... 

In this model system, as contrasted with the simple ferric ion reductase activity of the flavoprotein (38S), the metal is not the ultimate electron acceptor but presumably serves the dual role of oxygen activation and electron carrier. The reaction may involve superoxide anion since it is inhibited by superoxide dismutase (erthrocuprein) (394). Xanthine plus xanthine oxidase can also serve as electron donor, and this latter model system is also inhibited by superoxide dismutase (5P5). Superoxide dismutase also inhibits the menadione-mediated NADPH oxidase activity of NADPH-cytochrome P-450 (396) as well as the reconstituted benz-phetamine bydroxylation system (397). The involvement of NADPH-cytochrome P-450 reductase in microsomal lipid peroxidation has been confirmed by the demonstration that the reaction in microsomes is totally inhibited by antibody to tbe purified reductase (374). It has been suggested that lipid peroxidation by microsomes requires another component, in addition to the reductase, which takes the place of the ferric ion chelate in the model system (57. ). 

A separate study has shown that PhNHNHPh is an effective reaction mimic for the flavin cofactors in xanthine oxidases and cytochrome P-450 reductases, and in combination with O2 yields HOOH. 

One may mention the relative lack of information on the possible toxicity mechanisms of other groups of explosives. The administration of hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX 30-300 mg kg-1 daily for 13 weeks) to rats caused hypotriglycidire-mia, convulsions, and death [4], In contrast, pentaerythritol tetranitrate (PETN 0.5%-1.0% in standard diet for 13 weeks) was nontoxic to rats [80], RDX was much less cytotoxic to V79 and TK-6 mammalian cell cultures than TNT [9], There also are very few data on their reactions with mammalian enzymes. Rabbit liver cytochrome P-450 2B4 (EC 1.14.14.1) converted RDX into 4-nitro-2,4-diazabutanal, two nitrite ions, ammonium, and formaldehyde, consuming one equivalent NADPH [81]. However, it is unclear whether this slow reaction (kcat < 0.01 s-1) may contribute to the toxicity of RDX. Xanthine oxidase transformed octohydro-l,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) at a lower rate, 10.5 nmol h 1 mg 1 protein under anaerobic conditions, into nitrite, formaldehyde, nitrous oxide, formic acid, and ammonium [82], Our preliminary observations show that RDX was much less reactive substrate for P-450R and E. cloacae NR than NTO or ANTA [53], Thus, the mechanisms underlying toxicity of RDX remain undisclosed. 

Following the method of McCord and Fridovich (2), the superoxide ion is generated through the oxidation of xanthine by oxygen, a reaction catalyzed by xanthine oxidase as shown in Fig. 13. O2 then reacts with oxidized cytochrome c and it is transformed into Oj. The disappearance of superoxide is monitored spectrophotometrically through the increase of the absorption band at 550 nm, as a consequence of... 

Oxidations are the most common biotransformation reactions that occur with most drugs. There are several classes of enzymes that carry out these reactions cytochrome P450s, flavin monooxygenases, monoamine oxidases, xanthine oxidase, aldehyde oxidases, aldehyde dehydrogenases, and peroxidases. Typical reactions and substrate substructures for each of these classes of enzymes will be described. 

The first indication of an essential metabolic role for molybdenum was obtained in 1953, when it was discovered that xanthine oxidase, important in purine metabolism, was a metalloenzyme containing molybdenum. Subsequently the element was shown to be a component of two other enzymes, aldehyde oxidase and sulphite oxidase. The biological functions of molybdenum, apart from its reactions with copper (see p. 123), are concerned with the formation and activities of these three enzymes. In addition to being a component of xanthine oxidase, molybdenum participates in the reaction of the enzyme with cytochrome C and also facilitates the reduction of cytochrome C by aldehyde oxidase. 

The organization of xanthine oxidase appears to be quite complex. There is evidence that various substrates are not bound at the same site, and that the primary reaction of different substrates may occur with various ones of the cofactors. The oxidation of purines and aldehydes is inhibited by pteridyl aldehyde and by cyanide, but these reagents do not affect the oxidation of DPNH. It is possible that these inhibitors influence substrate binding sites and primary electron transport, respectively, and that the oxidation of DPNH involves a different binding site and avoids the cyanide-sensitive electron transport mechanism, which may well involve iron. Xanthine oxidase, and probably all flavoproteins, require —SH groups, but a definite function for these groups cannot be ascribed at this time. Similarly, various factors influence the reactions with oxidants differentially. Cyanide inhibits cytochrome reduction, but not the reactions with 0 or dyes. Reduction of either cytochrome c or nitrate depends upon the presence of molybdenum. These observations... 

---