Original Contribution INACTIVATION OF HUMAN Cu,Zn SUPEROXIDE DISMUTASE BY PEROXYNITRITE AND FORMATION OF HISTIDINYL RADICAL BEATRIZ ALVAREZ,* ,y VERO ´ NICA DEMICHELI,* ,y,z ROSARIO Dura ´n, § MADIA TRUJILLO, y,z CARLOS CERVEN ˜ ANSKY , § BRUCE A. FREEMAN, b and RAFAEL RADI y,z *Laboratorio de Enzimologı ´a, Facultad de Ciencias, Universidad de la Repu ´blica, 11400 Montevideo, Uruguay; z Departamento de Bioquı ´mica, Facultad de Medicina, Universidad de la Repu ´blica, 11800 Montevideo, Uruguay; y Center for Free Radical and Biomedical Research, Uruguay; § Instituto de Investigaciones Biolo ´gicas Clemente Estable 11600 Montevideo, Uruguay; and b Departments of Anesthesiology, Biochemistry, and Molecular Genetics, and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA (Received 13 January 2004; Revised 26 May 2004; Accepted 4 June 2004) Available online 25 June 2004 Abstract—Human recombinant copper – zinc superoxide dismutase (CuZnSOD) was inactivated by peroxynitrite, the product of the reaction between nitric oxide and superoxide. The concentration of peroxynitrite that decreased the activity by 50% (IC 50 ) was f 100 AM at 5 AM CuZnSOD and the inactivation was higher at alkaline pH. Stopped-flow determinations showed that the second-order rate constant for the direct reaction of peroxynitrite with CuZnSOD was (9.4 F 1.0)  10 3 M 1 s 1 per monomer at pH 7.5 and 37jC. Addition of peroxynitrite (1 mM) to CuZnSOD (0.5 mM) in the presence of the spin trap 2-methyl-2-nitrosopropane led to the electron paramagnetic resonance detection of an anisotropic signal typical of a protein radical adduct. Treatment with Pronase revealed a nearly isotropic signal consistent with the formation of histidinyl radical. The effects of nitrite, hydrogen peroxide, bicarbonate, and mannitol on the inactivation were assessed. Considering the mechanism accepted for the reaction of CuZnSOD with hydrogen peroxide and the fact that CuZnSOD promotes the nitration of phenolics by peroxynitrite, we herein propose that peroxynitrite reacts with CuZnSOD leading to nitrogen dioxide plus a copper-bound hydroxyl radical species that reacts with histidine residues, forming histidinyl radical. D 2004 Elsevier Inc. All rights reserved. Keywords—Peroxynitrite, Superoxide dismutase, Histidinyl radical, Superoxide, Nitric oxide, Free radicals INTRODUCTION The enzyme copper –zinc superoxide dismutase (CuZn- SOD) catalyzes the disproportionation of superoxide anion to dioxygen and hydrogen peroxide [1]. This antioxidant enzyme is present in the cytosol and mito- chondrial intermembrane space of eukaryotic cells and in the periplasmic space of bacterial cells as a homo- dimer of 32 kDa. Each monomer binds one copper and one zinc ion. The reaction mechanism involves the sequential reduction and reoxidation of Cu(II), for which both reactions are relatively pH-independent and pro- ceed with rate constants of 2  10 9 M 1 s 1 : O S 2 þ CuðIIÞZnSOD ! O 2 þ CuðIÞZnSOD; O S 2 þ CuðIÞZnSOD þ 2H þ ! H 2 O 2 þ CuðIIÞZnSOD: The Cu(II) ion is bound to four histidine residues. One of them also coordinates the Zn(II) ion, together with two more histidines and one aspartate. Several point mutations of CuZnSOD have been implicated in the devastating motor neuron disease familial amyotrophic lateral sclerosis (ALS) [2]. It is generally accepted that the disease is initiated by the gain of a new and toxic property [3]. Candidates for Address correspondence to: Beatriz Alvarez, Laboratorio de Enzimo- logı ´a, Facultad de Ciencias, Igua ´ 4225, 11400 Montevideo, Uruguay; Fax: +5982 5250749; E-mail: Beatriz.Alvarez@fcien.edu.uy; or Rafael Radi, Departamento de Bioquı ´mica, Facultad de Medicina, Avda. Gral. Flores 2125, 11800 Montevideo, Uruguay; Fax: +5982 9249563; E-mail: rradi@fmed.edu.uy. Free Radical Biology & Medicine, Vol. 37, No. 6, pp. 813 –822, 2004 Copyright D 2004 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter doi:10.1016/j.freeradbiomed.2004.06.006 813