Caratterizzazione biochimica e studi di stabilità proteica riguardanti forme mutate della Rieske ferredossina da Archea termifilica

Rieske proteins and Rieske ferredoxins are present in the three domains of life and are involved in a variety of cellular processes. Despite their functional diversity, these small Fe¿S proteins contain a highly conserved all-β fold, which harbors a [2Fe¿2S] Rieske center. Recently has been identify a novel subtype of Rieske ferredoxins presents in hyperthermophilic archea. The Acidianus ambivalens, Rieske ferredoxin 2 (RFd2) present a novel disulfide bridge within the Rieske fold that can be selectively reduced. When the disulfide is reduced has been observed a decrease in protein stability and an increase in the redox potential of the [2Fe2S] cluster. A salt bridge (due to the Glu49) situated near the prostetic group, seem to be the stronger in the protein has been individuated analyzing the structure of the protein. To investigate the role of the disulfide and of the salt bridge, three mutants were express: RFd2-C78A, RFd2-C74A, RFd2-E49A. This report describes the study of the role of a disulfide and of a salt bridge in the stability of the RFd2. RFd2 and its mutants were analyzed by spectroscopic techniques: Fluorescence, CD, UV-Vis absorption and EPR. The results demonstrate that mutants present a decrease in the stability in comparison to the WT (at pH 7, ΔTmWT-E49A= 3.13 ºC; ΔTmWT-C78A= 6.52 ºC).We observed that the trend of the melting temperature vs pH is different for the three proteins; this behaviour seem to be correlated with the variation of the cluster pKa between WT and mutants, measured by CD pH titration. It has been recently demonstrated that the pKa of the Rieske cluster is due to a sequential dissociation of protons from the Nε of the histidine that bind the [2Fe2S] cluster. Starting form that assumption we built a model that try to demonstrate the above correlation. Further results were obtained studying the unfolding kinetics of the proteins. The measured kinetics rate constants suggest a different kinetics for the unfolding of the tertiary structure and for the disintegration of the cluster. We observed that the RFd2-C78A presents a different behaviour in the kinetics of disintegration of the cluster, respect on RFd2 and RFd2-E49A. All the obtained results suggest that the disulfide and the salt bridge have an effect on the stability of the cluster. We also try to crystallize the RFd2-C78A. Small crystals has been obtained, which dimension is not sufficient to observe diffraction.