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Temperature-Induced Structural Changes in Putidaredoxin: A Circular Dichroism and UV-VIS Absorption Study



Vytautas Reipa, Marcia J. Holden, M P. Mayhew, V L. Vilker


Putidaredoxin (Pdx) is an 11,400 Da iron-sulfur protein that sequentially transfers two electrons to the cytochrome P450cam during the enzymatic cycle of the stereo specific camphor hydroxylation. We report two transitions in the Pdx UV-VIS absorption and CD temperature dependencies, occurring at 16.3 C 0.5 C and 28.4 C 0.5 C. The 16.3 C transition is attributed to the disruption of the hydrogen bonding between the active center bridging sulfur atom and Aspartate 38. The transition at 28.4 C occurs exclusively in the Pdxox at very nearly the same temperature as the earlier reported biphasicity in the redox potential. The formal potential temperature slope constancy reflects the relative stability of the concentration ratio of both oxidation states. The lower temperature transition affects both Pdx43d and Pdxox to a comparable extent, and their concentration ratio is constant. In contrast, the 28.4 C transition preferentially destabilizes Pdxox thereby accelerating the formal potential negative shift and lower redox reaction entropy. There is evidence to suggest that disrupting hydrogen bonding between iron ligating cysteines 45, 39 and residues threonine 47, serine 44 causes the 28.4 C transition. The sensitivity of the UV-VIS absorption and CD spectroscopy to subtle structural protein backbone transitions is demonstrated.
Biochimica Et Biophysica ACTA-Proteins and Proteomics
No. 1-2


circular dichroism, formal potential, hydrogen bonding, iron-surfur proteins, Putidaredoxin, UV-VIS spectroscopy


Reipa, V. , Holden, M. , Mayhew, M. and Vilker, V. (2004), Temperature-Induced Structural Changes in Putidaredoxin: A Circular Dichroism and UV-VIS Absorption Study, Biochimica Et Biophysica ACTA-Proteins and Proteomics (Accessed April 24, 2024)
Created June 1, 2004, Updated February 19, 2017