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

Oxidation-reduction midpoint potentials were determined, as a function of pH, for the disulfide/dithiol couples of spinach and pea thioredoxins <i>f</i>, for spinach and <i>Chlamydomonas reinhardtii</i> thioredoxins <i>m</i>, for spinach ferredoxin:thioredoxin reductase (FTR), and for two enzymes regulated by thioredoxin <i>f</i>, spinach phosphoribulokinase (PRK) and the fructose-1,6-bisphosphatases (FBPase) from pea and spinach. Midpoint oxidation-reduction potential (<i>E</i>_m) values at pH 7.0 of −290 mV for both spinach and pea thioredoxin <i>f</i>, −300 mV for both <i>C. reinhardtii</i> and spinach thioredoxin <i>m</i>, −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 ≥ 7.0, but with two components being present at pH values <7.0. The slopes of plots of <i>E</i>_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 <i>f</i> and <i>m</i>, for FTR, and for pea FBPase. The slope of the <i>E</i>_m versus pH profile for PRK shows three regions, consistent with the presence of p<i>K</i>_a values for the two regulatory cysteines in the region between pH 7.5 and 9.0.