The kinetics of inhibition of dopamine (3,4-dihydroxyphenethylamine) beta-mono-oxygenase by cyanide (CN-) and azide (N3-) ions have been investigated by using steady-state methods. Both anions show complex non-competitive-inhibition patterns with respect to ascorbate, suggestive of anion binding at two different sites on the oxidized enzyme. To further investigate this finding, e.p.r. titrations of CN- and N3- binding to the 63Cu-reconstituted enzyme were carried out. Addition of approx. 2 equiv. of CN- to copper elicits a new signal with g = 2.217, g = 2.025, A = 17.0 mT characteristic of a copper (II)-cyano complex. Simulations show that this signal accounts for half the copper (II) in the enzyme. The remainder of the enzyme-bound copper is expressed by a signal close to, but not identical with, that of native enzyme. Further addition of CN- induces a simultaneous decrease in intensity of both of these signals so that their 1:1 ratio is maintained. Binding of N3-, on the other hand, changes the e.p.r. spectrum to a form different from either that of the native or CN- -treated enzyme, and integrates to 100% of the copper in the enzyme (g = 2.252, g = 2.050, A = 16.5 mT). Resolved superhyperfine structure is apparent in the g region. N3- binding is also accompanied by the appearance of a broad charge-transfer band centred at 387 nm. Neither 9 nor 35 GHz e.p.r. spectra show evidence for more than one (non-interacting) species of Cu(II) in native enzyme and N3- derivatives. The binding and reactivity of CN-, on the other hand, argues against independent copper sites in the enzyme.

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1. Ascorbic acid deficiency results in a reduction in the activity of the hepatic mixed function oxidase systems in the guinea-pig. In this study, male Dunkin-Hartley guinea-pigs were given 0, 50, 100, 200 or 300 mg ascorbic acid/d in two equal doses in buffered sucrose solution (200 g/l) for 4 d. Controls received an equal volume of sucrose solution.2. A dose of 50 mg ascorbic acid/d resulted in a significant rise in the specific activities of both cytochromes P-450 and b,. At doses of 200 and 300mg ascorbic acid/d the concentration of both haemoproteins was significantly lower than the control values. These effects were mirrored by total microsomal haem concentration. 3. These results suggest that when given in large doses, ascorbic acid ceases to act simply as a vitamin and should be considered a drug competing for substrates and cofactors with co-administered drugs and endogenous substrates such as cholesterol.