Ternary protein complex of ferredoxin, ferredoxin:thioredoxin reductase, and thioredoxin studied by paramagnetic NMR spectroscopy.
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.
Xu
Xingfu
X
Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
Schürmann
Peter
P
Chung
Jung-Sung
JS
Hass
Mathias A S
MA
Kim
Sung-Kun
SK
Hirasawa
Masakazu
M
Tripathy
Jatindra N
JN
Knaff
David B
DB
Ubbink
Marcellus
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005288:Ferredoxins / Q000737:chemistry / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009068:Movement; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D012996:Solutions; D018724:Spinacia oleracea; D046939:Synechocystis / Q000201:enzymology; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism*
D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D010940:Plant Proteins; D012996:Solutions; D013879:Thioredoxins; D010088:Oxidoreductases; C021588:ferredoxin-thioredoxin reductase