A Spectroscopic Study of the Temperature Induced Modifications on Ferredoxin Folding and Iron−Sulfur Moieties†
- Smilja Todorovic ,
- Sónia S. Leal ,
- Carlos A. Salgueiro ,
- Ingo Zebger ,
- Peter Hildebrandt ,
- Daniel H. Murgida , and
- Cláudio M. Gomes
Abstract
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 α-helical content at relatively low temperatures (
∼ 44 °C), followed by the disruption of both iron−sulfur clusters (
∼ 53−60 °C). The degradation of the metal centers triggers major structural changes on the protein matrix, including the loss of tertiary contacts (
∼ 58 °C) and a change, rather than a significant net loss, of secondary structure (
∼ 60 °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.
†
The work was supported by Grants POCTI/BIO/43105 (to D.H.M.) and POCTI/QUI/37521 and POCTI/QUI/45758 (to C.M.G.) from the Fundação Ciência e Tecnologia (FCT/MCES, Portugal). S.S.L. is a recipient of a Ph.D. fellowship (SFRH/BD/18653/2004) from Fundação para a Ciência e Tecnologia (FCT/MCES, Portugal).
‡
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa.
§
These authors have equally contributed to the work.
‖
Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa. Previous address: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
⊥
Technische Universität Berlin.
*
C.M.G.: [email protected]; tel, +351214469332; fax, +351214411277. D.H.M.: [email protected]; tel, +541145763378 ext. 124; fax, +541145763341.
#
Universidad de Buenos Aires.
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