Oxidation-Reduction Properties of Chloroplast Thioredoxins, Ferredoxin:Thioredoxin Reductase, and Thioredoxin f-Regulated Enzymes

Abstract

Oxidation-reduction midpoint potentials were determined, as a function of pH, for the disulfide/dithiol couples of spinach and pea thioredoxins f, for spinach and Chlamydomonas reinhardtii thioredoxins m, for spinach ferredoxin:thioredoxin reductase (FTR), and for two enzymes regulated by thioredoxin f, spinach phosphoribulokinase (PRK) and the fructose-1,6-bisphosphatases (FBPase) from pea and spinach. Midpoint oxidation-reduction potential (E m ) values at pH 7.0 of -290 mV for both spinach and pea thioredoxin f, -300 mV for both C. reinhardtii and spinach thioredoxin m, -320 mV for spinach FTR, -290 mV for spinach PRK, -315 mV for pea FBPase, and -330 mV for spinach FBPase were obtained. With the exception of spinach FBPase, titrations showed a single two-electron component at all pH values tested. Spinach FBPase exhibited a more complicated behavior, with a single two-electron component being observed at pH values g 7.0, but with two components being present at pH values <7.0. The slopes of plots of E m versus pH were close to the -60 mV/pH unit value expected for a process that involves the uptake of two protons per two electrons (i.e., the reduction of a disulfide to two fully protonated thiols) for thioredoxins f and m, for FTR, and for pea FBPase. The slope of the E m versus pH profile for PRK shows three regions, consistent with the presence of pK a values for the two regulatory cysteines in the region between pH 7.5 and 9.0.The ferredoxin/thioredoxin system of oxygenic photosynthetic organisms plays an important role in the regulation of the carbon metabolism of these organisms (1-3). The initial step in the thioredoxin regulatory pathway, which has been extensively characterized in spinach and pea chloroplasts, involves the reduction of ferredoxin:thioredoxin reductase (hereafter abbreviated FTR 1 ) by the reduced ferredoxin generated during light-driven noncyclic electron flow (1-3). Spinach leaf FTR, the best characterized of these enzymes, is a 25.6 kDa heterodimeric protein located in the chloroplast stroma (1-3). FTR contains a unique cluster that serves to stabilize the one-electron-reduced intermediate formed after the first electron donation by ferredoxin, during the two-electron reduction of the activesite disulfide of the oxidized enzyme to the two cysteine thiols present in reduced FTR (4, 5). FTR reduces thioredoxin in a reaction in which the two cysteines at the active site of the reduced enzyme become oxidized to a cystine disulfide, while the active-site disulfide of the oxidized thioredoxin becomes reduced to two cysteine thiols (1-5). FTR reduces both of the thioredoxins found in chloroplasts, thioredoxin f and thioredoxin m (monomeric proteins with molecular masses of ∼12 kDa that contain a conserved -WCGPCactive site), with equal efficiency. However, the two chloroplast thioredoxins display differential but overlapping reactivities among the array of identified target proteins (1-3). Although regulatory reduction by thioredoxin m appears to be restricted to glucose-6-phosph...

Repression of photosynthesis gene expression by formation of a disulfide bond in CrtJ

“…The data were plotted and fit to a two-electron Nernst curve by using SIGMA PLOT software with maximum fluorescence set at the most reduced sample. E m values obtained were independent of time, over times ranging from 0.5 to 2.0 h and independent of total glutathione concentration, over a range from 2.5 to 10.0 mM, as would be expected if true redox equilibrium has been attained at all E h values (13)(14)(15).…”

“…A measurement of relative percent disulfide bond formation at various redox potentials was measured by assaying fluorescence emission of CrtJ when incubated in the presence of monobromobimane (mBBr). Previous studies (13)(14)(15) have indicted that mBBr fluorescence increases significantly when it forms a covalent linkage with a Cys-sulfhydral. Thus, the level of mBBr fluorescence provides a good measurement of free sulfhydrals versus disulfides that are present in protein preparations that have been equilibrated in buffers containing different redox potentials.…”

α-Glucan, water dikinase (GWD): A plastidic enzyme with redox-regulated and coordinated catalytic activity and binding affinity

“…Enzyme samples were incubated under aerobic conditions at 25°C in activation mixtures containing varying amounts of oxidized and reduced DTT to achieve equilibrium at defined redox potentials. Activation mixtures consisted of 50 l of solution containing 1.2 g of GWD, 100 mM Hepes-KOH (pH 7.9), catalytic amounts of Trx f (0.1 M), and 80 mM total DTT to ensure a stable redox poise (25,26). Samples were allowed to equilibrate at ambient potentials (E h ) defined by different ratios of reduced:oxidized DTT for 2 h. Subsequently, GWD activity was measured at 25°C and pH 7.9 over 30 min.…”

Thioredoxin–thioredoxin reductase – a system that has come of age

“…These proteins function as NADH-dependent reductants of peroxyredoxins that in turn reduce alkyl hydroperoxides. The final Minireview will cover this interesting system [10].The chloroplast thioredoxins f and m, that are reduced by ferredoxin±thioredoxin reductase rather than by thioredoxin reductase are mentioned briefly in the first Minireview [11]. Biochem.…”

“…The chloroplast thioredoxins f and m, that are reduced by ferredoxin±thioredoxin reductase rather than by thioredoxin reductase are mentioned briefly in the first Minireview [11]. Biochem.…”