Ternary Protein Complex of Ferredoxin, Ferredoxin:Thioredoxin Reductase, and Thioredoxin Studied by Paramagnetic NMR Spectroscopy

• Published in: Journal of the American Chemical Society 2009-12, Vol.131 (48), p.17576-17582
• Main Authors: Xu, Xingfu, Schürmann, Peter, Chung, Jung-Sung, Hass, Mathias A. S, Kim, Sung-Kun, Hirasawa, Masakazu, Tripathy, Jatindra N, Knaff, David B, Ubbink, Marcellus
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Catalytic Domain; Crystallography, X-Ray; Ferredoxins - chemistry; Ferredoxins - metabolism; Iron-Sulfur Proteins - chemistry; Iron-Sulfur Proteins - metabolism; Magnetic Resonance Spectroscopy; Models, Molecular; Movement; Oxidoreductases - chemistry; Oxidoreductases - metabolism; Plant Proteins - chemistry; Plant Proteins - metabolism; Protein Binding; Solutions; Spinacia oleracea; Synechocystis - enzymology; Thioredoxins - chemistry; Thioredoxins - metabolism
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja904205k

In oxygenic photosynthetic cells, carbon metabolism is regulated by a light-dependent redox signaling pathway through which the light signal is transmitted in the form of electrons via a redox chain comprising ferredoxin (Fd), ferredoxin:thioredoxin reductase (FTR), and thioredoxin (Trx). Trx affects the activity of a variety of enzymes via dithiol oxidation and reduction reactions. FTR reduces an intramolecular disulfide bridge of Trx, and Trx reduction involves a transient cross-link with FTR. NMR spectroscopy was used to investigate the interaction of Fd, FTR, and an m-type Trx. NMR titration experiments indicate that FTR uses distinct sites to bind Fd and Trx simultaneously to form a noncovalent ternary complex. The orientation of Trx-m relative to FTR was determined from the intermolecular paramagnetic broadening caused by the [4Fe-4S] cluster of FTR. Two models of the noncovalent binary complex of FTR/Trx-m based on the paramagnetic distance restraints were obtained. The models suggest that either a modest or major rotational movement of Trx must take place when the noncovalent binary complex proceeds to the covalent complex. This study demonstrates the complementarity of paramagnetic NMR and X-ray diffraction of crystals in the elucidation of dynamics in a transient protein complex.