UNIVERSITY OF MALTA LIFE SCIENCE RESEARCH SEMINARS Web: http://www.um.edu.mt/events/scisem/ Email: scisem@um.edu.mt Abstract form Title: Metal Selectivity and Specificity of Iron Superoxide Dismutase from Escherichia coli. Presenter: Diane Farrugia Contact address: Department of Physiology and Biochemistry, University of Malta, MSD2080. Tel: Fax: Email: dfar0012@um.edu.mt Presentation date: 1st July 2013 . Abstract The requirement of oxygen for aerobically living species necessitates protective mechanisms that remove potentially damaging reactive oxygen species (ROS). Superoxide dismutases (SODs) are important antioxidant metalloenzymes which protect the essential cell structures from the superoxide radical by catalysing their dismutation to oxygen and hydrogen peroxide. There are four classes of SODs, grouped according to the central metal ion (Fe, Mn, Cu/Zn, and Ni) at the active site. FeSOD and MnSOD share extensive sequence homology rendering them nearly superimposable, with an identical metal binding site. These two proteins can accommodate either Mn or Fe at their active sites, but are found to be highly selective and specific for the “correct” metal. They are inactive when the “incorrect” metal ion is artificially inserted into the active site. During this research project, an Escherichia coli FeSOD mutant, prepared by cloning and site-directed mutagenesis in our lab by Prof. G. J. Hunter and Dr. T. Hunter was studied. This mutant protein, FeSOD[dblDGF], contains five amino acid changes in its sequence to mimic those in MnSOD. The aim of this research was therefore to obtain further knowledge with regards to selectivity for the metal and specificity of the reaction. Metal selectivity was investigated through metal replacement both in vivo and in vitro, and a method was developed. Physicochemical properties were analysed to determine metal content (ICP-MS, MP-AES), enzyme activity and thus specificity (spectrophotometry, zymography) and structural integrity (circular dichrosim, X-Ray crystallography).