Study of manganese (II) centers from different types of Superoxide Dismutases by High-Field EPR spectroscopy

TABARES, L C; UN S.; CORTEZ, N.

Rosario (Argentina)

Congreso; XXXV Reunión Anual de la Sociedad Argentina de Biofísica SAB; 2006

Sociedad Argentina de Biofísica SAB

Superoxide dismutases (SODs) are proteins involved in the defense against oxidative stress conditions. Enzymes from the Fe/Mn family exhibit a mononuclear metal-site with a trigonal bipyramidal geometry. Although the tridimensional structure is highly conserved among all the members of this family, the metal exchange normally results in a non-active protein. Using high-field electron paramagnetic resonance (HFEPR, 285GHz) spectroscopy we have investigated the Mn(II) electronic structure of five different SODs. Although these proteins are considered to be structural homologues these studies showed that they exhibited differences in their zero-field interaction. Moreover, the total zero-field interaction of the protein low activity with Fe showed the highest values, while the spectra of the two protein highly active with Fe resembled one each other The effect of pH and substrate analogues on the Mn(II) HFEPR spectra of these five SODs were investigated. Azide was able to coordinate directly to the Mn(II) site of enzymes from E. coli and from R. capsulatus, although no direct binding was observed for the other proteins studied. Fluoride affected the spectra of all SODs, but no evidence for six coordinated Mn(II) was found in any case. High pH only induced changes in the HFEPR spectrum of the Mn-substituted FeSOD from E. coli for which a pKa of 9.8 was estimated. Except the direct binding of azide, all the other effect appeared to correspond to a subtle perturbation metal site due to the modification on the second coordination-sphere. All these outer-sphere effects appear to occur at the level of Tyr34 which has been proposed as responsible for the pH dependence in Fe(II) and Mn(III) SODs centers. Our work in MnSODs illustrates how sensitive HFEPR spectroscopy is as probe of Mn(II) centers. This technique not only provide a straightforward way to discriminate between five and six-coordinated Mn(II)-center in proteins, but is also able to detect subtle changes of the zero-field interaction.