ELSEVIER Bioeleetroehemistry and Bioenergefics 38 (1995) 397-400 Short communication A polarographic study of the catalytic mechanism of the iron-containing superoxide dismutase from Escherichia coli Emanuele Argese *, Emilio F. Orsega, Catia Granito, Ligia M. Moretto Faculty of Sciences, Universit~ Ca' Foscari, Dorsoduro 2137, 30173 Venice, Italy Received 17 November 1994; in revised form 26 January 1995 Abstract The dependence of the activity of iron-containing superoxide dismutase from Escherichia coli on pH and ionic strength was investigated extensively in the ranges 7.7-11.0 and 0.02-0.25 M respectively. The results show that the enzymatic activity diminishes with both increasing pH and increasing ionic strength, reaching a minimum value at about pH 11. The activity decrease with increasing pH was ascribed to a gradual titration, by OH- ion, of the acid group Fe3+-H20. This gives rise to a deprotonated enzymatic form with a residual activity which is about 10% that of the protonated form. The dependence of the activity on ionic strength was interpreted as a Debye-HUckel effect. Keywords: Iron superoxide dismutase; Catalytic polarographic waves; Catalytic mechanism; Escherichia coli 1. Introduction The superoxide dismutases (SODs) are ubiquitous metal-proteins which enhance the rate of spontaneous dis- mutation of superoxide ion (0 2) into 02 and H202 by some orders of magnitude [1], according to the general scheme 202 +2H + SOD~H202+O 2 (1) The catalytic action of these metal-enzymes is very important in protecting the cells from injury by free radi- cals coming from the monovalent reduction of molecular oxygen in the redox cellular processes [2,3]. The SODs known up to now are classified in three types according to the metal ion: copper-zinc, manganese and iron containing superoxide dismutase, shortened to Cu,Zn-SOD, Mn-SOD and Fe-SOD respectively. Cu,Zn- SOD is mainly present in eucaryotic organisms, Mn-SOD is present in eucaryotes and in evolved prokaryotes, while Fe-SOD is found in primitive species of prokaryotes [3,4]. Cu,Zn-SODs extracted from several types of mammals and plants have been the subject of many investigations of their structure and catalytic mechanism, whereas studies of Mn-SOD and Fe-SOD are comparatively few. In particu- * Corresponding author. 0302-4598/95/$09.50 © 1995 Elsevier Science S.A. All rights reserved SSDI 0302-4598(95)01815-8 lar, some aspects of the catalytic mechanism and of the kinetic features of Fe-SOD are not completely known. Fe-SOD generally consists of two identical subunits, each of molecular weight of about 19 000 and each appar- ently binding a single Fe 3+ ion. X-ray crystallographic analyses have shown that the metal binding site contains three histidine residues and one aspartate residue. A water molecule can occupy a fifth coordination position. In the resting enzyme, iron is pre- sent in the trivalent oxidation state [5]. NMR relaxation and spectroscopic studies [6] suggested the presence of an ionizable group on or near Fe 3+ in the pH range 9-10. Investigations of the kinetic properties of Fe-SOD, car- ded out by the stopped-flow spectrophotometric method [7] and by pulse radiolysis [8], showed that Fe-SOD from Escherichia coli catalyzes the 02 dismutation with a mechanism exhibiting saturation kinetics [9]. The Fe- SOD-catalyzed reaction rate can be represented, according to Michaelis-Menten formalism, by the relationship [10] d[O21 kv [Fe-SOD] [O2 ] dt kM + [02] (2) In a previous work [11] the suitability of the polaro- graphic method of the catalytic currents for measuring Fe-SOD activity was demonstrated. The activity is deter-