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 superoxide dismutase in the oxidized and reduced form and to the copper-free derivative has been investigated by Fourier transform infrared spectroscopy. The antisymmetric stretching band of the azide is shifted to higher energy upon coordination to the copper atom of the oxidized form of the native enzyme. Similar spectral changes occur upon interaction of the anion with the Cu-diethylenetriamine model compound. On the other hand, interaction of azide with the native reduced form of the enzyme results in a band shift toward lower energy with respect to the free anion band. The same shift is observed after reaction of the azide with free lysine or arginine but not when it is reacted with other amino acid residues. The antisymmetric band of the azide is not perturbed by addition of the reduced arginine-modified enzyme; it is likely shifted toward higher energy upon addition of oxidized arginine-modified enzyme while it is again shifted toward lower energy in the presence of the copper-free derivative of the unmodified enzyme. It is concluded that azide does not directly coordinate to the copper in the reduced form of Cu,Zn superoxide dismutase but it remains in the active-site pocket in electrostatic interaction with the guanidinium group of Arg141, which is an invariant residue in this class of enzymes.
Leone
M
M
Institute of Physics and Istituto Nazionale di Fisica della Materia, University of Palermo, 90123 Palermo, Italy. leone@fisica.unipa.it
Cupane
A
A
Militello
V
V
Stroppolo
M E
ME
Desideri
A
A
D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002417:Cattle; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D010084:Oxidation-Reduction; D011073:Polyamines / Q000737:chemistry; D019810:Sodium Azide / Q000737:chemistry / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D013482:Superoxide Dismutase / Q000737:chemistry / Q000378:metabolism*
D004791:Enzyme Inhibitors; D011073:Polyamines; C005391:diethylenetriamine; D003300:Copper; D001120:Arginine; D019810:Sodium Azide; D013482:Superoxide Dismutase; D008239:Lysine