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

Abstract

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.

A continuous-wave electron–nuclear double resonance (X-band) study of the Cu2+ sites of particulate methane mono-oxygenase of Methylococcus capsulatus (strain M) in membrane and pure dopamine β-mono-oxygenase of the adrenal medulla

“…Protein-model complex * The line-width was 4.5, 4.5, 5.0 mT and the line-shape was 40 % Gaussian, 60 % Lorentzian; EPR parameters from [19]. † The line-width is 1.0, 1.0, 6.5 mT and 100 % Gaussian line-shape; the present study.…”

“…The EPR spectrum of DβM shows significant differences upon addition of the inhibitors azide and cyanide [19], whereas the EPR spectrum of the copper site of pMMO is unaffected by these small molecules. Here, we suggest that they do not bind to the EPR visible copper site of pMMO.…”

“…The temperature-dependence of the microwave power saturation behaviour suggests that the two coppers are not magnetically interacting, indicating that they are at least 7 A H apart [18], in agreement with the crystal structure of PAMcc, in which a distance of 11 A H was found between the two coppers. EPR, ESEEM, EXAFS and Fouriertransform infrared (' FTIR ') studies on DβM have shown that the two mononuclear copper sites can be distinguished by using copper-binding ligands, such as azide or cyanide [19,20]. From these investigations, it was concluded that each Cu# + centre is ligated by two or three histidine ligands and one or two O\N ligands [17,21].…”

Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion

“…After observing that FGE contained ∼1 copper atom/enzyme, we made an effort to remove the metal from the protein to demonstrate its requirement for catalytic activity. Cyanide is known to be an inhibitor of copper-containing oxidases ( 36 – 38 ). Attempting to extract copper through pretreatment with 5 molar eq of KCN did decrease the activity of Hs -cFGE (but not Sc -FGE) by a modest amount, suggesting that it could gain access to the bound copper ( Fig.…”

“…We observed that the catalytic rates of both human and bacterial FGE were significantly increased upon activation with stoichiometric amounts of Cu 2+ , and that turnover was inhibited by the addition of cyanide to FGE reaction mixtures. Previous reports have shown that the inhibition of aerobic copper oxidases by CN − results from ligation of CN − at the metal center (in the reduced state of Cu 1+ ), which prevents oxygen binding and substrate activation ( 38 ). Furthermore, FGE activity was extinguished completely after treating the enzyme with a combination of a chelator and a reductant, reinforcing the highly specific and tight binding interaction between FGE and Cu 2+ .…”