Kinetic and e.p.r. studies of cyanide and azide binding to the copper sites of dopamine (3,4-dihydroxyphenethylamine) β-mono-oxygenase

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Abstract
The kinetics of inhibition of dopamine .beta.-monooxygenase by cyanide (CN-) and azide (N3-) ions were investigated by using steady-state methods. Both anions show complex non-competitive-inhibition patterns with respect to ascorbate, suggestive of anion binding at 2 different sites on the oxidized enzyme. To further investigate this finding, EPR titrations of CN- and N3- binding to the 63Cu-reconstituted enzyme were carried out. Addition of .apprx. 2 equiv. of CN- to Cu elicits a new signal with g.dblvert. = 2.217, g.perp. = 2.025, A.dblvert. = 17.0 mT [millitesla] characteristic of a Cu (II)-cyano complex. Simulations show that this signal accounts for half the Cu (II) in the enzyme. The remainder of the enzyme-bound CU 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 these signals so that their 1:1 ratio is maintained. Binding of N3- changes the EPR spectrum to a form different from either that of the native or CN- treated enzyme, and integrates to 100% of the Cu in the enzyme (g.dblvert. = 2.252, g.perp. = 2.050, A.dblvert. = 16.5 mT). Resolved superhyperfine structure is apparent in the g.perp. region. N3- binding is also accompanied by the appearance of a broad charge-transfer band centered at 387 nm. Neither 9 nor 35 GHz EPR spectra show evidence for more than 1 (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 Cu sites in the enzyme.