Kinetic and e.p.r. studies of cyanide and azide binding to the copper sites of dopamine (3,4-dihydroxyphenethylamine) beta-mono-oxygenase.
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.
D001205:Ascorbic Acid / Q000378:metabolism; D001386:Azides / Q000494:pharmacology*; D001665:Binding Sites; D003300:Copper; D003486:Cyanides / Q000494:pharmacology*; D004299:Dopamine beta-Hydroxylase / Q000037:antagonists & inhibitors*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D046911:Macromolecular Substances; D014439:Tyramine / Q000378:metabolism
D001386:Azides; D003486:Cyanides; D046911:Macromolecular Substances; D003300:Copper; D004299:Dopamine beta-Hydroxylase; D001205:Ascorbic Acid; D014439:Tyramine