ABSTRACT
The variable subunit of spinach ferredoxin:thioredoxin reductase (FTR) has an extended N-terminus compared to FTRs from other sources and this was proposed to contribute to the instability of the protein. We constructed two N-terminal truncation mutants of recombinant FTR by removing 16 or 24 residues from the variable subunit. The mutant proteins are readily expressed and show half-saturation values (S(0.5)) for ferredoxin and thioredoxin f comparable to WT. However, truncation increases significantly their stability. Using the stabilized FTR an exposed Cys on its thioredoxin contact surface could be substituted without altering its properties, whereas the replacement of an active site Cys by Ser completely destabilized the protein.
Subject(s)
Enzyme Stability/genetics , Oxidoreductases/genetics , Protein Subunits/genetics , Sequence Deletion , Spinacia oleracea/enzymology , Amino Acid Sequence , Amino Acid Substitution , Binding Sites/genetics , Iron-Sulfur Proteins , Kinetics , Mutagenesis, Site-Directed , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Sequence AlignmentABSTRACT
Ferredoxin:thioredoxin reductase (FTR) is a heterodimeric Fe&z.sbnd;S containing disulfide reductase involved in the light-dependent activation of photosynthetic enzymes. We have designed a dicistronic construct for the heterologous expression of this nucleus encoded chloroplast protein in Escherichia coli. The coding sequences for the two mature subunits have been inserted in tandem into the expression vector pET-3d. This dicistronic construct is correctly translated yielding soluble, perfectly functional FTR. The recombinant enzyme is composed of both subunits, contains the correctly inserted Fe&z.sbnd;S cluster as evidenced by its spectral properties and is indistinguishable from the enzyme isolated from leaves in its capacity to activate chloroplast fructose-1,6-bisphosphatase, one of the well known light activated enzymes of the Calvin cycle.
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 (Em) 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 >/= 7.0, but with two components being present at pH values <7.0. The slopes of plots of Em 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 Em versus pH profile for PRK shows three regions, consistent with the presence of pKa values for the two regulatory cysteines in the region between pH 7.5 and 9.0.
