Functional and molecular characterization of plastid terminal oxidase from rice (Oryza sativa) |
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Authors: | Qiuju Yu Kathleen Feilke Anja Krieger-Liszkay Peter Beyer |
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Affiliation: | 1. Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany;2. Commissariat à l''Energie Atomique (CEA) Saclay, iBiTec-S, CNRS UMR 8221, Service de Bioénergétique, Biologie Structurale et Mécanisme, 91191 Gif-sur-Yvette Cedex, France |
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Abstract: | The plastid terminal oxidase (PTOX) is a plastohydroquinone:oxygen oxidoreductase that shares structural similarities with alternative oxidases (AOX). Multiple roles have been attributed to PTOX, such as involvement in carotene desaturation, a safety valve function, participation in the processes of chlororespiration and setting the redox poise for cyclic electron transport. We have investigated a homogenously pure MBP fusion of PTOX. The protein forms a homo-tetrameric complex containing 2 Fe per monomer and is very specific for the plastoquinone head-group. The reaction kinetics were investigated in a soluble monophasic system using chemically reduced decyl-plastoquinone (DPQ) as the model substrate and, in addition, in a biphasic (liposomal) system in which DPQ was reduced with DT-diaphorase. While PTOX did not detectably produce reactive oxygen species in the monophasic system, their formation was observed by room temperature EPR in the biphasic system in a [DPQH2] and pH-dependent manner. This is probably the result of the higher concentration of DPQ achieved within the partial volume of the lipid bilayer and a higher Km observed with PTOX-membrane associates which is ≈ 47 mM compared to the monophasic system where a Km of ≈ 74 μM was determined. With liposomes and at the basic stromal pH of photosynthetically active chloroplasts, PTOX was antioxidant at low [DPQH2] gaining prooxidant properties with increasing quinol concentrations. It is concluded that in vivo, PTOX can act as a safety valve when the steady state [PQH2] is low while a certain amount of ROS is formed at high light intensities. |
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Keywords: | DPQ, decyl-plastoquione PTOX, plastid terminal oxidase nOG, n-octyl β-D-glucopyranoside DCPIP, 2,6-dichlorophenol-indophenol-Na IPTG, isopropyl β-D-1-thiogalactopyranoside BSA, bovine serum albumin DoDm, n-dodecyl β-D-maltoside DeDm, n-decyl β-D-maltoside CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate LDAO, N,N-dimethyldodecylamine N-oxide CMC, critical micelle concentration GPC, gel permeation chromatography SOD, superoxide dismutase |
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