FOURIER-TRANSFORM INFRARED-ANALYSIS OF THE INTERACTION OF AZIDE WITH THE ACTIVE-SITE OF OXIDIZED AND REDUCED BOVINE CU,ZN SUPEROXIDE-DISMUTASE

Binding of azide to the native and arginine-modified bovine Cu,Zn supe roxide dismutase in the oxidized and reduced form and to the copper-fr ee derivative has been investigated by Fourier transform infrared spec troscopy. The antisymmetric stretching band of the azide is shifted to higher energy upon coordination to the copper atom of the oxidized fo rm of the native enzyme. Similar spectral changes occur upon interacti on of the anion with the Cu-diethylenetriamine model compound. On the other hand, interaction of azide with the native reduced form of the e nzyme results in a band shift toward lower energy with respect to the free anion band. The same shift is observed after reaction of the azid e with free lysine or arginine but not when it is reacted with other a mino acid residues. The antisymmetric band of the azide is not perturb ed by addition of the reduced arginine-modified enzyme; it is likely s hifted toward higher energy upon addition of oxidized arginine-modifie d enzyme while it is again shifted toward lower energy in the presence of the copper-free derivative of the unmodified enzyme. It is conclud ed that azide does not directly coordinate to the copper in the reduce d form of Cu,Zn superoxide dismutase but it remains in the active-site pocket in electrostatic interaction with the guanidinium group of Arg 141, which is an invariant residue in this class of enzymes.