Structural and Biochemical Insights into the Reactivity of Thioredoxin h1 from Chlamydomonas reinhardtii
Christophe H. Marchand,
Simona Fermani,
Jacopo Rossi,
Libero Gurrieri,
Daniele Tedesco,
Julien Henri,
Francesca Sparla,
Paolo Trost,
Stéphane D. Lemaire,
Mirko Zaffagnini
Affiliations
Christophe H. Marchand
Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, Unité Mixte de Recherche 8226 CNRS Sorbonne Université, 13 rue Pierre et Marie Curie, 75005 Paris, France
Simona Fermani
Department of Chemistry “Giacomo Ciamician”, University of Bologna, via Selmi 2, 40126 Bologna, Italy
Jacopo Rossi
Laboratory of Molecular Plant Physiology, Department of Pharmacy and Biotechnology, University of Bologna, via Irnerio 42, 40126 Bologna, Italy
Libero Gurrieri
Laboratory of Molecular Plant Physiology, Department of Pharmacy and Biotechnology, University of Bologna, via Irnerio 42, 40126 Bologna, Italy
Daniele Tedesco
Bio-Pharmaceutical Analysis Section (Bio-PhASe), Department of Pharmacy and Biotechnology, University of Bologna, via Belmeloro 6, 40126 Bologna, Italy
Julien Henri
Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, Unité Mixte de Recherche 8226 CNRS Sorbonne Université, 13 rue Pierre et Marie Curie, 75005 Paris, France
Francesca Sparla
Laboratory of Molecular Plant Physiology, Department of Pharmacy and Biotechnology, University of Bologna, via Irnerio 42, 40126 Bologna, Italy
Paolo Trost
Laboratory of Molecular Plant Physiology, Department of Pharmacy and Biotechnology, University of Bologna, via Irnerio 42, 40126 Bologna, Italy
Stéphane D. Lemaire
Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, Unité Mixte de Recherche 8226 CNRS Sorbonne Université, 13 rue Pierre et Marie Curie, 75005 Paris, France
Mirko Zaffagnini
Laboratory of Molecular Plant Physiology, Department of Pharmacy and Biotechnology, University of Bologna, via Irnerio 42, 40126 Bologna, Italy
Thioredoxins (TRXs) are major protein disulfide reductases of the cell. Their redox activity relies on a conserved Trp-Cys-(Gly/Pro)-Pro-Cys active site bearing two cysteine (Cys) residues that can be found either as free thiols (reduced TRXs) or linked together by a disulfide bond (oxidized TRXs) during the catalytic cycle. Their reactivity is crucial for TRX activity, and depends on the active site microenvironment. Here, we solved and compared the 3D structure of reduced and oxidized TRX h1 from Chlamydomonas reinhardtii (CrTRXh1). The three-dimensional structure was also determined for mutants of each active site Cys. Structural alignments of CrTRXh1 with other structurally solved plant TRXs showed a common spatial fold, despite the low sequence identity. Structural analyses of CrTRXh1 revealed that the protein adopts an identical conformation independently from its redox state. Treatment with iodoacetamide (IAM), a Cys alkylating agent, resulted in a rapid and pH-dependent inactivation of CrTRXh1. Starting from fully reduced CrTRXh1, we determined the acid dissociation constant (pKa) of each active site Cys by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analyses coupled to differential IAM-based alkylation. Based on the diversity of catalytic Cys deprotonation states, the mechanisms and structural features underlying disulfide redox activity are discussed.
