Characterization of ferredoxin:thioredoxin reductase modified by site-directed mutagenesis.

作者: Dominique A. Glauser , Florence Bourquin , Wanda Manieri , Peter Schürmann

DOI: 10.1074/JBC.M313851200

关键词: ChemistryBiochemistryMutantActive siteSite-directed mutagenesisWild typeThioredoxinFerredoxin-thioredoxin reductaseFerredoxinThioredoxin reductase

摘要: Ferredoxin:thioredoxin reductase (FTR) is a key regulatory enzyme of oxygenic photosynthetic cells involved in the reductive regulation important target enzymes. It catalyzes two-electron reduction disulfide thioredoxins with electrons from ferredoxin involving 4Fe-4S cluster and an adjacent active-site disulfide. We replaced Cys-57, Cys-87, His-86 active site Synechocystis FTR by site-directed mutagenesis studied properties mutated proteins. Mutation either cysteines yields inactive enzymes, which have different spectral properties, indicating reduced Fe-S when inaccessible Cys-87 oxidized accessible Cys-57 replaced. The latter mutant can be reversibly dithionite showing that it functional. C57S very stable protein, whereas C87A more labile because missing interaction cluster. replacement greatly reduces its catalytic activity supporting proposal increases nucleophilicity neighboring cysteine. Ferredoxin forms non-covalent complexes wild type (WT) FTRs, are except mutant. WT FTRs form covalent heteroduplexes modified thioredoxins. In particular, formed represent interesting one-electron-reduced reaction intermediates, split Heteroduplexes demonstrating ability to simultaneously dock thioredoxin ferredoxin, accord proposed mechanism structural analyses.

参考文章(25)
Peter Schürmann, [28] Ferredoxin: Thioredoxin system Methods in Enzymology. ,vol. 252, pp. 274- 283 ,(1995) , 10.1016/0076-6879(95)52030-9
Peter Schürmann, Ferredoxin-dependent thioredoxin reductase: a unique iron-sulfur protein. Methods in Enzymology. ,vol. 347, pp. 403- 411 ,(2002) , 10.1016/S0076-6879(02)47040-1
Ana Chueca, Mariam Sahrawy, Eduardo A. Pagano, Julio López Gorgé, Chloroplast fructose-1,6-bisphosphatase: structure and function. Photosynthesis Research. ,vol. 74, pp. 235- 249 ,(2002) , 10.1023/A:1021243110495
A.F.S.A. Habeeb, [37] Reaction of protein sulfhydryl groups with Ellman's reagent Methods in Enzymology. ,vol. 25, pp. 457- 464 ,(1972) , 10.1016/S0076-6879(72)25041-8
Hans-Peter Klenk, Rebecca A. Clayton, Jean-Francois Tomb, Owen White, Karen E. Nelson, Karen A. Ketchum, Robert J. Dodson, Michelle Gwinn, Erin K. Hickey, Jeremy D. Peterson, Delwood L. Richardson, Anthony R. Kerlavage, David E. Graham, Nikos C. Kyrpides, Robert D. Fleischmann, John Quackenbush, Norman H. Lee, Granger G. Sutton, Steven Gill, Ewen F. Kirkness, Brian A. Dougherty, Keith McKenney, Mark D. Adams, Brendan Loftus, Scott Peterson, Claudia I. Reich, Leslie K. McNeil, Jonathan H. Badger, Anna Glodek, Lixin Zhou, Ross Overbeek, Jeannine D. Gocayne, Janice F. Weidman, Lisa McDonald, Teresa Utterback, Matthew D. Cotton, Tracy Spriggs, Patricia Artiach, Brian P. Kaine, Sean M. Sykes, Paul W. Sadow, Kurt P. D'Andrea, Cheryl Bowman, Claire Fujii, Stacey A. Garland, Tanya M. Mason, Gary J. Olsen, Claire M. Fraser, Hamilton O. Smith, Carl R. Woese, J. Craig Venter, The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus Nature. ,vol. 390, pp. 364- 370 ,(1997) , 10.1038/37052
D R Smith, L A Doucette-Stamm, C Deloughery, H Lee, J Dubois, T Aldredge, R Bashirzadeh, D Blakely, R Cook, K Gilbert, D Harrison, L Hoang, P Keagle, W Lumm, B Pothier, D Qiu, R Spadafora, R Vicaire, Y Wang, J Wierzbowski, R Gibson, N Jiwani, A Caruso, D Bush, J N Reeve, Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics. Journal of Bacteriology. ,vol. 179, pp. 7135- 7155 ,(1997) , 10.1128/JB.179.22.7135-7155.1997
Peter Sch�rmann, Anne Lise Stritt-Etter, Junsheng Li, Reduction of ferredoxin:thioredoxin reductase by artificial electron donors. Photosynthesis Research. ,vol. 46, pp. 309- 312 ,(1995) , 10.1007/BF00020445
W. Manieri, L. Franchini, L. Raeber, S. Dai, A.-L. Stritt-Etter, P. Schürmann, N-terminal truncation of the variable subunit stabilizes spinach ferredoxin:thioredoxin reductase FEBS Letters. ,vol. 549, pp. 167- 170 ,(2003) , 10.1016/S0014-5793(03)00811-1
Masakazu Hirasawa, Michel Droux, Kevin A. Gray, J.Milton Boyer, Dan J. Davis, Bob B. Buchanan, David B. Knaff, Ferredoxin-thioredoxin reductase: Properties of its complex with ferredoxin Biochimica et Biophysica Acta (BBA) - Bioenergetics. ,vol. 935, pp. 1- 8 ,(1988) , 10.1016/0005-2728(88)90101-6
Shaodong Dai, Cristina Schwendtmayer, Peter Schürmann, S Ramaswamy, Hans Eklund, Redox signaling in chloroplasts: cleavage of disulfides by an iron-sulfur cluster Science. ,vol. 287, pp. 655- 658 ,(2000) , 10.1126/SCIENCE.287.5453.655