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...