DPNH oxidase

P-hydroxylase of mitochondria which converts DOGA to corticosterone, and which requires TPNH as a reducing cosubstrate. DPNH and ascorbic acid are ineffective in this reaction, but together with the microsomal preparation containing the ascorbic acid-dependent DPNH-oxidase described below, they will replace TPNH. 

Staudinger s interpretation of this involves the central problem of such hydroxylation reactions the generation of free radicals from O2 as the possible hydroxylating agents (K4). The hypothesis is that the reaction of TPNH with O2 in Ae mitochondrial 11-P-hydroxylase generates such free radicals, and so does the oxidation of ascorbic acid by the DPNH-oxidase-cytochrome be system (Eq. 15). The monodehydroascorbic acid formed in the latter system is already well documented. 

Ascorbic Acid-dependent DPNH-Oxidase Microsomes of adrenal medullas contain an enzyme which oxidizes DPNH but not TPNH, and like the similar enzyme from plant tissues (K2, N2) utilizes oxidized ascorbic acid but not dehydroascorbic acid as the acceptor. Complexed Fe + in ferricyanide or cytochrome c can also serve... 

K2. Kem, M., and Racker, E., Activation of DPNH oxidase by an oxidation product of ascorbic acid. Arch. Biochem. Biophys. 48, 235-236 (1954). 

From evidence at hand, therefore. Fraction I is specifically involved in phenylalanine hydroxylation and may form oxygen complexes. Fraction II is a widely distributed ferroprotein, probably a DPNH oxidase which can utilize oxygen itself as an electron acceptor... 

From the present point of view, either Fraction I-O2 or Fraction I-O is a specific substrate for a DPNH oxidase, if the hydroxyl-ation mechanism is primarily Type II or Type III. The hydroxyl-ating intermediate would then be either Fraction I-O or Fraction I-Oj, respectively (equations, 89, 90, and 91). The whole system... 

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... 

Aldehyde Oxidase. Besides xanthine oxidase, liver contains a more specific aldehyde oxidase. This enzyme does not oxidize reduced DPN or purines, but does convert a large number of aliphatic and aromatic aldehydes to the corresponding acids. This enzyme contains molybdenum and an iron protoporphyrin in addition to FAD. It is completely reduced by aldehydes, whereas aldehydes bleach xanthine oxidase only partially (compared with hydrosulfite). Aldehyde dehydrogenase oxidizes DPNH at no more than 1 per cent of the rate of aldehyde oxidation. Cytochrome c, oxygen, and dyes all serve as oxidants. 

DPN is the electron acceptor for three, and TPN for one of the five oxidases of the citric-acid cycle. The nature of the primary electron acceptor for succinate is still unknown. The reaction of DPNH and TPNH with one of the cytochromes (c) is known to be mediated by reductases containing riboflavin in their prosthetic groups. Both DPN and TPN cytochrome reductases have been isolated in homogeneous state from an-... 

Xanthine oxidase —oxidation of aldehydes, purines, and DPNH. 

The enzyme catalyzing conversion of 17a-hydroxyprogesterone to 17a,21-hydroxyprogesterone occurs in a system comprisii the combined supernatant and microsomal fractions of beef adrenals (594,632,633). Enzymic 21-hydroxylation requires oxyg, and is activated by nicotinamide, DPN, and ATP (594,632). These requirements are similar to those which were observed with crude systems catalyzing llj8-hydroxylation (see above), and to the liver phenylalanine hydroxylase (556,557,770) which has been separated into two fractions, and which is activated by DPNH. The preliminary characterization of 21-hydroxylase therefore suggests that it is a mixed fimction oxidase. 

The properties of mixed function oxidases are summarized in Table XVII. These enzyme systems oxidize both aromatic and aliphatic substances with consumption of oxygen. TPNH, DPNH, dihydroxy-fumarate, and o-diphenols serve as electron donors for reduction of one atom of the oxygen molecule. In general, metal-binding reagents inhibit enzymes of this class iron, copper, and possibly manganese form prosthetic groups. 

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