The structure and function of the mitochondrial peroxiredoxin SP-22

Gourlay, Louise Jane (2003) The structure and function of the mitochondrial peroxiredoxin SP-22. PhD thesis, University of Glasgow.

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Substrate protein-22 (SP-22) is a 22kDa bovine protein that functions as a thioredoxin-dependent peroxidase in the mitochondrial matrix. The exact physiological targets of SP-22 remain to be elucidated; however its antioxidant activity has been well documented in the protection of several free radical- sensitive enzymes. SP-22 has been assigned to a novel peroxidase family called the peroxiredoxins (Prxs). Prxs use electrons provided by free thiol groups to reduce hydrogen peroxide, a potent oxidising agent that can contribute to oxidative stress either directly or via the hydroxyl radical generated by the Fenton reaction. Prxs can be divided into two sub-groups (1-Cys and 2-Cys) depending on the number of conserved cysteine residues they possess. SP-22 is a 2-Cys Prx with three cysteines, not an uncommon feature, at positions 47, 66 and 168. Following sequence analysis C47 is proposed to be the catalytic residue, containing a sulphenic acid group, functioning as a 2-electron redox centre. The cloning and overexpression of recombinant SP-22 and three cysteine mutants (C47S, C66S and C168S) in bacterial cells as N-terminal His-tag proteins is reported. The overexpression conditions for optimal protein solubility and the subsequent purification by metal chelate chromatography were also determined. A comparison of reducing and non reducing SDS-PAGE of wild type and mutant SP-22s established that SP-22 contains a dimeric unit linked by two intermolecular disulphide bonds, in its oxidised state. These involve C47 of one monomer and C168 of the opposing subunit. This result is consistent with findings for other 2-Cys Prx members. Previous work in this laboratory involving the purification of the mitochondrial 2-oxoacid dehydrogenase member, pyruvate dehydrogenase (PDC) from bovine heart, and its subsequent separation into its three enzyme components by size exclusion chromatography, established that the dihydrolipoamide dehydrogenase component (E3) co-eluted with another contaminating protein. Following N-terminal sequencing this protein was identified to be SP-22. Preliminary results using the techniques of Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) have confirmed a physical association between SP-22 and E3, binding with an affinity in the micromolar range. The functional significance of this interaction is discussed with respect to the possible protection of PDC and the oxoglutarate dehydrogenase complex (OGDC) against oxidative modification of the catalytically-active thiol groups possessed by the individual components. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biochemistry.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Lindsay, Prof. J. Gordon
Date of Award: 2003
Depositing User: Enlighten Team
Unique ID: glathesis:2003-71166
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 05 Aug 2022 16:27
Thesis DOI: 10.5525/gla.thesis.71166

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