Effects of Metal Ligation and Oxygen on the Reversibility of the Thermal Denaturation of Pseudomonas Aeruginosa Azurin

Published

January 22, 2002

Author(s)

A Sandberg, J Leckner, Y. Shi, Frederick P. Schwarz, B G. Karlsson

Abstract

Thermodynamic equilibrium transition models in DSC are only applicable to reversible processes, but reversibility of the thermal transitions of proteins is comparatively rare because of intermolecular aggregation of denatured proteins and the degradation that occurs at high temperatures. The cupredoxin azurin from Pseudomonas aeruginosa has previously been found to exhibit irreversible thermal denaturation, both as holo- and apoproteins [Engeseth, H.R., and McMillin, D.R. (1986) Biochemistry 25, 2448-2455]. In this study, however, we demonstrate that this Β-barrel protein of Greek key topology in fact unfolds reversibly in anaerobic solutions when nonreducible metal ions are ligated to the protein. We show that it is the metal-coordinating cysteine residue (C112) that becomes exclusively oxidized in a transition metal catalyzed oxidation reaction with dissolved O₂ at high temperatures. Both Cu(I)- and Zn(II)-coordinating wild-type azurin therefore unfold reversibly in anaerobic solutions, as well as 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-state transitions. Unfolding of Cu(II)-coordinating azurin is, on the other hand, always irreversible due to autoxidation of the thiolate resulting in Cu(I) and a thiyl radical prone to oxidation.

Citation

Biochemistry

Volume

41

Issue

3

Pub Type

Journals

Keywords

azurin, differential scanning calorimetry, mutants, proteins, thermal stability, thermodynamics, two-state transitions

Citation

Sandberg, A. , Leckner, J. , Shi, Y. , Schwarz, F. and Karlsson, B. (2002), Effects of Metal Ligation and Oxygen on the Reversibility of the Thermal Denaturation of Pseudomonas Aeruginosa Azurin, Biochemistry (Accessed July 21, 2024)