The prominent conformational plasticity of lactoperoxidase: a chemical and pH stability analysis.
Lactoperoxidase (LPO) is a structurally complex and stable mammalian redox enzyme. Here we aim at evaluating the influence of ionic interactions and how these intertwine with the structural dynamics, stability and activity of LPO. In this respect, we have compared LPO guanidinium hydrochloride (GdmCl) and urea denaturation pathways and performed a detailed investigation on the effects of pH on the LPO conformational dynamics and stability. Our experimental findings using far-UV CD, Trp fluorescence emission and ESR spectroscopies clearly indicate that LPO charged-denaturation with GdmCl induced a sharp two-step process versus a three-step unfolding mechanism induced by urea. This differential effect between GdmCl and urea suggests that ionic interactions must play a rather prominent role in the stabilization of LPO. With both denaturants, the protein core was shown to retain activity up to near the respective C(m) values. Moreover, a pH titration of LPO evidenced no significant conformational alterations or perturbation of heme activity within the 4 to 11 pH interval. In contrast, alterations of ionic interactions by poising LPO at pH 3, 2 and 12 resulted in a loss of secondary structure, loosening of tertiary contacts and loss of activity, which appear to be associated with the perturbation of the hydrophobic core, as evidenced by ANS binding, as well as disruption of the heme pocket demonstrated by optical and EPR spectroscopies. Overall, LPO is characterised by a high degree of peripheral structural plasticity without perturbation of the core heme moiety. The possible physiological meaning of such features is discussed.
Boscolo
Barbara
B
Dipartimento di Chimica I.F.M., Università di Torino, Torino, Italy.
Leal
Sónia S
SS
Salgueiro
Carlos A
CA
Ghibaudi
Elena M
EM
Gomes
Cláudio M
CM
D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D006418:Heme / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007784:Lactoperoxidase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D055672:Static Electricity
D006418:Heme; D007784:Lactoperoxidase