Thermodynamics of the Dimer−Decamer Transition of Reduced Human and Plant 2-Cys Peroxiredoxin

Abstract

Isothermal titration calorimetry (ITC) is a powerful technique for investigating self-association processes of protein complexes and was expected to reveal quantitative data on peroxiredoxin oligomerization by directly measuring the thermodynamic parameters of dimer-dimer interaction. Recombinant classical 2-cysteine peroxoredoxins from Homo sapiens, Arabidopsis thaliana, and Pisum sativum as well as a carboxy-terminally truncated variant were subjected to ITC analysis by stepwise injection into the reaction vessel under various redox conditions. The direct measurement of the decamer-dimer equilibrium of reduced peroxiredoxin revealed a critical concentration in the very low micromolar range. The data suggest a cooperative assembly above this critical transition concentration where a nucleus facilitates assembly. The rather abrupt transition indicates that assembly processes do not occur below the critical transition concentration while oligomerization is efficiently triggered above it. The magnitude of the measured enthalpy confirmed the endothermic nature of the peroxiredoxin oligomerization. Heterocomplexes between peroxiredoxin polypeptides from different species were not formed. We conclude that a functional constraint conserved the dimer-decamer transition with highly similar critical transition concentrations despite emerging sequence variation during evolution.

Kinetic analysis of structural influences on the susceptibility of peroxiredoxins 2 and 3 to hyperoxidation

“…For all redox states, the dissociation of oligomers into dimeric units occurred much more readily for Prx3 than Prx2. As expected [18,27,28], the reduced forms of both proteins were oligomeric under our SEC conditions. However, in contrast with Prx2, the oxidized (disulfide) form of Prx3 and the reduced protein at a lower concentration were fully dissociated.…”

“…However, hyperoxidized Prx3 with one disulfide and one hyperoxidized active site was dimeric (Figure 7B). Lowering the pH to 7 stabilizes the decameric structure of Prx2 [27,29]. This pH effect was also observed with Prx3, with the disulfide and the hyperoxidized dimers associating to dodecamers at pH 7 (result not shown).…”

“…The catalytic units of 2-cysteine Prxs function as stable homodimers, which associate to form non-covalent oligomers [24,25]. Studies of the relationship between redox state and oligomerization have shown that for various Prxs including Prx2 [26,27], the reduced form is most stable as a decamer, the disulfide more readily dissociates into dimers and the decameric state of fully hyperoxidized protein is even more stable. There is less known for Prx3 [18,28] and the quaternary structure of the hyperoxidized dimers has not been investigated.…”

Novel Regulators in Photosynthetic Redox Control of Plant Metabolism and Gene Expression

“…it is not clear whether FTR is able to reduce all Trxs or how Trx-z as integral component of the transcriptional complex is reduced. The interactions between the network components need to be qualitatively and quantitatively assessed by in vitro and in vivo methods such as isothermal titration calorimetry, kinetic assays, and fluorescence resonance energy transfer similar as done for the chloroplast 2-CysPrx (Barranco-Medina et al, 2008;Muthuramalingam et al, 2009). Models of partial networks and simulations of electron fluxes and redox states will help to test our knowledge and predict regulatory states that can be validated in further experiments (Kemp et al, 2008).…”

The Dual-Targeted Plant Sulfiredoxin Retroreduces the Sulfinic Form of Atypical Mitochondrial Peroxiredoxin

“…After an initial 60-s delay, series of 1.6 mL of protein solution were injected into the cell at 3-min intervals to reach equilibrium after each injection. The first injection was an incomplete injection of 1 mL and was ignored during data analysis (Barranco-Medina et al, 2008a). Data were analyzed using the software provided by MicroCal Systems (ORIGIN).…”