Metal replacement and structural analysis of iron superoxide dismutase from Escherichia coli

Abstract

Superoxide dismutases are claimed to be the most important antioxidant proteins in all living organisms, serving as the first line of defence against superoxide radicals and in essence oxidative stress (Ken et aI., 2005) by catalytically converting the superoxide. radical to oxygen and hydrogen peroxide at near diffusion controlled rates (Abreu & Cabelli, 2010). The structurally homologous iron (FeSOD) and manganese superoxide dismutases (MnSOD) can accommodate either Mn or Fe at their active sites, but they tend to show selectivity toward their 'correct' metal ion and reaction is highly specific, showing inactivity with the 'incorrect' metal ion.

The mutant protein FeSOD[Q69G/A141Q/G142D/A68GIY76F] (FeSOD[dblDGFD was previously prepared by Dr. T. Hunter and Prof. G. J. Hunter. This is an FeSOD with five amino acid residues substituted to their corresponding MnSOD amino acids, with the intention oftuming an FeSOD into a MnSOD. This research project therefore aimed at substituting the metal at the active site using in vivo and in vitro techniques and to study the metal substituted protein using physicochemical analysis.

In vivo methods were improved so that FeSOD and FeSOD[ dblDGF] incorporated minimal iron at the active site. SOD production in the presence of GroESL, during culturing of E. coli OX326A cells in minimal media, was substantially less. Metal content analysis showed that although this chaperone increased manganese levels in FeSOD, it had the opposite effect on FeSOD[dblDGF]. Activity was retained even at very low quantities of the 'correct' metal. An in vitro substitution method was developed throughout this research that allowed the complete removal of metal from the active site, producing the apo-SOD. This protein was subsequently metallated with the metal of interest, giving reliable results.

FeSOD[dblDGF] appear to be very selective towards iron at the active site, similar to FeSOD. Activity of this mutant was observed to be just 25 % when compared to the wild type. When substituted with manganese no detectable activity was present, showing that this combination of mutant residues does not make FeSOD behave as a MnSOD.

Although activity with manganese at the active site was not obtained, thermostability results from CD analysis showed that this mutant protein is thermostable. Both the iron containing and the manganese substituted proteins were found to have a similar transition temperature (Tm) but lower than that of the wild type FeSOD. The electron density map of this mutant showed structural similarities to that of MnSOD, as active site residues showed similar conformation, with some bond length measurement more closely related to MnSOD than to FeSOD. EPR spectra comparisons of FeSOD and FeSOD[dblDGF] also confirmed that the electronic configuration of the active site environment is actually different between these two proteins.