Ferredoxin thioredoxin reductase

Staples CR, Gaymard E, Stritt-Etter A-L, et al. 1998. Role of the 6484] cluster in mediating disulfide reduction in spinach ferredoxin thioredoxin reductase. Biochemistry 37 4612-20. 

Droux, M., Jacquot, J.P., Miginac-Maslow, M., Gadal, P., Huet, J.C., Crawford, N.A., Yee, B.C., and Buchanan, B.B. 1987. Ferredoxin-thioredoxin reductase, an iron-sulfur enzyme linking light to enzyme regulation in oxygenic photosynthesis purification and properties of the enzyme from C3, C4, and cyanobacterial species. Arch. Biochem. Biophys. 252, 426-439. 

Hirasawa, M., Droux, M., Gray, K.A., Boyer, J.M., Davis, D.J., Buchanan, B.B. and Knaff, D.B. 1988. Ferredoxin-thioredoxin reductase properties of its complex with ferredoxin. Biochim. Biophys. Acta 935, 1-8. 

Jacquot, J.P., Stein, M., Suzuki, A., Liottet, S., Sandoz, G. and Miginiac-Maslow, M. 1997. Residue Glu-91 of Chlamydomonas reinhardtii ferredoxin is essential for electron transfer to ferredoxin-thioredoxin reductase. FEES Lett. 400, 293-296. 

Figure 8 Proposed catalytic mechanism for ferredoxin-thioredoxin reductase...

In chloroplasts, oxidized thioredoxin is reduced by ferredoxin in a reaction catalyzed by ferredoxin-thioredoxin reductase. This enzyme contains a 4Fe-4S cluster that couples two one-electron oxidations of reduced ferredoxin to the two-electron reduction of thioredoxin. Thus, the activities of the light and dark reactions ofphotosynthesis are coordinated through electron transfer from reduced ferredoxin to thioredoxin and then to component enzymes containing regulatory disulfide bonds (Figure 20.16). We shall return to thioredoxin when we consider the reduction of ribonucleotides (Section 25.3). 

The ferredoxin-thioredoxin system. Thioredoxins are small proteins that transfer electrons from reduced ferredoxin to certain enzymes (Figure 13.17). (Recall that ferredoxin is an electron donor in PSI.) When exposed to light, PSI reduces ferredoxin, which then reduces ferredoxin-thioredoxin reductase (FTR),... 

Using the light energy captured by PSI, energized electrons are donated to ferredoxin. Electrons donated by ferredoxin to FTR (ferredoxin-thioredoxin reductase) are used to reduce the disulfide bridge of thioredoxin. Thioredoxin then reduces the disulfide bridges of susceptible enzymes. Some enzymes are activated by this process, whereas others are inactivated. 

Corn leaf NADP- malate dehydrogenase (NADP- MDH), spinach ferredoxin, ferredoxin thioredoxin reductase and thioredoxin m were purified to homogeneity as described earlier (1). Pig ad enal ferredoxin was prepared as in (1). Human lymphocyte thioredoxin was cloned, overexpressed and purified from E coli as described in (2). 

Light activation of NADP- MDH The activation mecium (100 (il) comprised the following components 100 imol Tris HCI pH 8.0, 10 (imol ferredoxin, 10 iimol thioredoxin, 2 (imol ferredoxin thioredoxin reductase,. 7 (imol NADP- MDH, 100 units catalase and pea thylakoids equivalent to 25 ig chlorophyll. When indcated, ferredoxin and thioredoxin were replaced by their animal counterparts (ADX and HTR). Photoactivation vras performed undw saturating white light and nitrogen. At appropriate times, aliquots were removed and used fa the spectrophotometric detamination of activity as in (1). 

The light signal, produced by the thylakoids, is transmitted via several proteins ferredoxin, ferredoxin-thioredoxin reductase (FTR) and thioredoxins to the target enzymes. 

The different proteins spinach ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) and thioredoxin m (TRX) and corn NADP-MDH were purified to homogeneity by already published procedures (4,5). EDTA-NaCI washed thylakoids were used to avoid contamination by stromal proteins.The thylakoids were pretreated with high light (4000 pMol quanta.m-2.sec-1) for different lengths of time. All the proteins of the system and NADP-MDH were then added, and MDH activated for a few min under moderate light (300 Mol quanta.m-2.sec-1). The reconstituted system comprised, in 100 pi total volume 100 mM Tris buffer, pH 7.9, thylakoids (20 pg chi), 10 pM Fd, 2 pM FTR, 10 pM TRX and 2.5 pM MDH. MDH activity and the thiol content of the different proteins of the mixture were then measured. Enzyme activity was measured by the decrease in absorbance at 340 nm in the presence of NADPH and oxaioacetate. Protein thiol quantitation was done by derivatization with 14C-labelled iodoacetate or iodoacetamide (6) and either TCA-precipitation of all the proteins, or HPLC separation of each of them. A special separation procedure was devised to recover the proteins from the reaction medium. It was done by molecular size exclusion on a TSK 3000 HPLC column and allowed a quantitative recovery of each of the proteins. The use of iodoacetamide as derivatizing agent and the addition of 300 mM NaCI to the elution buffer were necessary to obtain an efficient separation (7). 

Spinach ferredoxin, thioredoxin m, ferredoxin-thioredoxin-reductase and com NADP-MDH were purified as previously described (4). Light activation of NADP-MDH, labeling of thiols and purification of the labeled protein... 

FBPase undergoes a reductive activation by accepting electrons from ferredoxin through the ferredoxin-thioredoxin system, which works as a link between the photosynthetic electron transport and enzyme regulation (7). The existence of non-covalent interactions of FBPase with the chloroplast membrane could improve the efficiency of its reductive light-activation. In this context the strong FBPase-thioredoxin interaction (8), the association between ferredoxin, thioredoxin and ferredoxin-thioredoxin reductase (9), the binding of ferredoxin to thylakoid membranes (10), and the existence of membrane-bound thioredoxins (11) may be relevant. 

The latent ATPase activity of CF undergoes light activation in intact chloroplasts (Mills, Hind 1979). The activation depends on both AyH and thiol-reduction of CF. The latter is probably mediated by thioredoxin which undergoes photoreduction via ferredoxin-thioredoxin reductase, thus resembling the activation of a few carbon cycle enzymes (Buchanan 1980 ... 

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