Effects of Metal Ligation and Oxygen on the Reversibility of the Thermal Denaturation of Pseudomonas aeruginosa Azurin
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- 作者：Anders Sandberg ; Johan Leckner ; Ying Shi ; Frederick P. Schwarz ; and B. Gö ; ran Karlsson
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：January 22, 2002
- 年：2002
- 卷：41
- 期：3
- 页码：1060 - 1069
- 全文大小：141K
- 年卷期：v.41,no.3(January 22, 2002)
- ISSN：1520-4995
文摘
Thermodynamic equilibrium transition models in DSC are only applicable to reversibleprocesses, but reversibility of the thermal transitions of proteins is comparatively rare because ofintermolecular aggregation of denatured proteins and the degradation that occurs at high temperatures.The cupredoxin azurin from Pseudomonas aeruginosa has previously been found to exhibit irreversiblethermal denaturation, both as holo- and apoprotein [Engeseth, H. R., and McMillin, D. R. (1986)Biochemistry 25, 2448-2455]. In this study, however, we demonstrate that this 
-barrel protein of Greekkey topology in fact unfolds reversibly in anaerobic solutions when nonreducible metal ions are ligatedto the protein. We show that it is the metal-coordinating cysteine residue (C112) that becomes exclusivelyoxidized in a transition metal catalyzed oxidation reaction with dissolved O2 at high temperatures. BothCu(I)- and Zn(II)-coordinating wild-type azurin therefore unfold reversibly in anaerobic solutions, as wellas the Zn(II)-coordinating disulfide-deficient C3A/C26A mutant. Correspondingly, apoazurin mutantsC112A and C112S unfold reversibly, even in aerobic solutions, and exhibit nearly perfect two-statetransitions. Unfolding of Cu(II)-coordinating azurin is, on the other hand, always irreversible due toautoxidation of the thiolate resulting in Cu(I) and a thiyl radical prone to oxidation.
-barrel protein of Greekkey topology in fact unfolds reversibly in anaerobic solutions when nonreducible metal ions are ligatedto the protein. We show that it is the metal-coordinating cysteine residue (C112) that becomes exclusivelyoxidized in a transition metal catalyzed oxidation reaction with dissolved O2 at high temperatures. BothCu(I)- and Zn(II)-coordinating wild-type azurin therefore unfold reversibly in anaerobic solutions, as wellas the Zn(II)-coordinating disulfide-deficient C3A/C26A mutant. Correspondingly, apoazurin mutantsC112A and C112S unfold reversibly, even in aerobic solutions, and exhibit nearly perfect two-statetransitions. Unfolding of Cu(II)-coordinating azurin is, on the other hand, always irreversible due toautoxidation of the thiolate resulting in Cu(I) and a thiyl radical prone to oxidation.