A spectroscopic study of the temperature induced modifications on ferredoxin folding and iron-sulfur moieties.

Thermal perturbation of the dicluster ferredoxin from Acidianus ambivalens was investigated employing a toolbox of spectroscopic methods. FTIR and visible CD were used for assessing changes of the secondary structure and coarse alterations of the [3Fe4S] and [4Fe4S] cluster moieties, respectively. Fine details of the disassembly of the metal centers were revealed by paramagnetic NMR and resonance Raman spectroscopy. Overall, thermally induced unfolding of AaFd is initiated with the loss of -helical content at relatively low temperatures (T(app)(m) approximately 44 degrees C), followed by the disruption of both iron-sulfur clusters (T(app)(m) approximately 53-60 degrees C). The degradation of the metal centers triggers major structural changes on the protein matrix, including the loss of tertiary contacts (T(app)(m) approximately 58 degrees C) and a change, rather than a significant net loss, of secondary structure (T(app)(m) approximately 60 degrees C). This latter process triggers a secondary structure reorganization that is consistent with the formation of a molten globule state. The combined spectroscopic approach here reported illustrates how changes in the metalloprotein organization are intertwined with disassembly of the iron-sulfur centers, denoting the conformational interplay of the protein backbone with cofactors.