Molecular cloning, expression analysis and iron metal cofactor characterisation of a superoxide dismutase from Toxoplasma gondii.

A genomic region of 12 kb encompassing the gene encoding the superoxide dismutase (SOD) of Toxoplasma gondii has been cloned. The gene contains four exons of 121, 42, 381 and 59 bp which are separated by three introns of 321, 202, and 577 bp, respectively. The open reading frame can be translated into a protein of 201 amino acids with a molecular mass of 22.6 kDa. Alignment indicated that it is a FeSOD, a type only found in bacteria, protozoa and chloroplast of higher plants. Recombinant SOD was expressed in a Escherichia coli double mutant lacking both MnFeSOD and FeSODs. The presence of iron as metal cofactor was confirmed by measurements of iron by absorption mass spectrometry and electron paramagnetic resonance studies. Semi-quantitative reverse transcribed polymerase chain reaction experiments showed a similar amount of SOD transcripts in two developmental stages of T. gondii. Antibodies raised against the purified recombinant protein detected SOD protein in both bradyzoite and tachyzoite forms suggesting this SOD might be essential for the intracellular growth of both developmental stages. Southern blot analysis indicated that SOD occured as a single copy gene in T. gondii genome.

EPR and ligand field studies of iron superoxide dismutases and iron-substituted manganese superoxide dismutases: relationships between electronic structure of the active site and activity.

The problem of metal selectivity of iron/manganese superoxide dismutases (SODs) is addressed through the electronic structures of active sites using electron paramagnetic resonance and ligand field calculations. Studies of wild-type iron(III) SOD (FeSOD) from Escherichia coli and from Methanobacterium thermoautotrophicum and iron-substituted manganese(III) SOD (Fe(sub)MnSOD) from E. coli and from Serratia marcescens are reported. EPR spectroscopy of wild-type enzymes shows transitions within all three Kramers doublets identified by their g values. From the temperature dependence of the observed transitions, the zero-field splitting is found to be negative, D = -2 +/- 0.2 cm-1. The electronic structure is typical of a distorted trigonal bipyramid, all the EPR features being reproduced by ligand field analysis. This unique and necessary electronic structure characterizes wild-type enzymes whatever their classification from the amino acid sequence into iron or manganese types, as E. coli FeSOD or M. thermoautotrophicum FeSOD. In iron-substituted manganese SODs, reduced catalytic activity is found. We describe how inhomogeneity of all reported substituted MnSODs might explain the activity decrease. EPR spectra of substituted enzymes show several overlapping components. From simulation of these spectra, one component is identified which shares the same electronic structure of the wild-type FeSODs, with the proportion depending on pH. Ligand field calculations were performed to investigate distortions of the active site geometry which induce variation of the excitation energy of the lowest quartet state. The corresponding coupling between the ground state and the excited state is found to be maximum in the geometry of the native SODs. We conjecture that such coupling should be considered in the electron-transfer process and in the contribution of the typical electronic structure of FeSOD to the activity.

Paramagnetic NMR spectroscopy of native and cobalt substituted manganese superoxide dismutase from Escherichia coli.

Manganese containing superoxide dismutase from E. coli has been investigated through paramagnetic NMR spectroscopy. The spectrum of the native form was rationalized using a tau(s) = 3x10(-11) s for the Mn(III) ion, consistent with previous estimates from NMRD measurements. Mn(III) has been replaced by a Co(II) ion and a tentative assignment of the NMR spectrum of the Co(II)-substituted derivative has been proposed, based on T1, chemical shifts and 1D-NOE data. The metal coordination geometry is provided by three histidines and a carboxylate group. The presence of a solvent molecule as a loosely bound fifth ligand is also proposed. The NMR data of the Co(II)-substituted derivative of E. coli MnSOD differs from those of Co(II)SOD from other bacterial sources. This suggests that Co(II) substitution is an efficient method to address the problem of metal ion selectivity in superoxide dismutase.