TYR36PHE MUTATION IN MN(II) SUPEROXIDE DISMUTASES AVOID THE INTERACTION WITH SUBSTRATE ANALOGUES

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

Rosario (Argentina)

Congreso; XLII Reunión Anual de la Sociedad Argentina de investigación en Bioquímica y Biología Molecular SAIB; 2006

SAIB Sociedad Argentina de investigación en Bioquímica y Biología Molecular

Superoxide dismutases are proteins involved in the defense against oxidative stress conditions. They have a mononuclear site with a pentacoordinated metal ion (Mn or Fe). Using high-field electron paramagnetic resonance (HFEPR) spectroscopy we have investigated the Mn(II) site of the R. capsulatus protein and its dependence with temperature and addition of azide and fluoride. Azide was found to directly coordinate the Mn(II). The formation of this complex was characterized by a reduction in the zero-field splitting. Fluoride dramatically affected the HFEPR spectrum but no evidence for six coordinated centers was detected. After incubation of the protein at low temperatures temperature a distinct six-line component was detectable. The newly six-line signal was assigned to the movement of a water molecule which normally lies into the substrate access channel hydrogen bonded to the Tyr36. Tyr36 is part of the substrate access channel and when it was replaced by a Phe there was no detectable interactions with azide or fluoride. By contrast, the low-temperature equilibrium was displaced towards the hexacoordinated form. Based on these results we propose a two-states binding model for these interaction in which Tyr36 acts as bridge between both possible binding sites. This model could represent the mode in which superoxide interacts with the active site during the reductive half-reaction.R. capsulatus protein and its dependence with temperature and addition of azide and fluoride. Azide was found to directly coordinate the Mn(II). The formation of this complex was characterized by a reduction in the zero-field splitting. Fluoride dramatically affected the HFEPR spectrum but no evidence for six coordinated centers was detected. After incubation of the protein at low temperatures temperature a distinct six-line component was detectable. The newly six-line signal was assigned to the movement of a water molecule which normally lies into the substrate access channel hydrogen bonded to the Tyr36. Tyr36 is part of the substrate access channel and when it was replaced by a Phe there was no detectable interactions with azide or fluoride. By contrast, the low-temperature equilibrium was displaced towards the hexacoordinated form. Based on these results we propose a two-states binding model for these interaction in which Tyr36 acts as bridge between both possible binding sites. This model could represent the mode in which superoxide interacts with the active site during the reductive half-reaction.