Effect of pH on the oxidation-reduction properties of thioredoxins.
Oxidation-reduction midpoint potential (E(m)) versus pH profiles were measured for wild-type thioredoxins from Escherichia coli and from the green alga Chlamydomonas reinhardtii and for a number of site-directed mutants of these two thioredoxins. These profiles all exhibit slopes of approximately -59 mV per pH unit, characteristic of the uptake of two protons per reduction of an active-site thioredoxin disulfide, at acidic, neutral, and moderately alkaline pH values. At higher pH values, these profiles exhibit slopes of either -29.5 mV per pH unit, characteristic of the uptake of one proton per disulfide reduced, or are pH-independent, indicating that neither proton uptake nor proton release is associated with reduction of the active-site disulfide. Reduction of the two wild-type thioredoxins is accompanied by the uptake of two protons even at pH values where the more acidic cysteine thiol group of the reduced proteins would be expected to be completely unprotonated. The effect of site-directed mutagenesis of two highly conserved aspartate residues that play important structural and/or catalytic roles in both thioredoxins, and which could in principle play a role in proton transfer, on the pK(a) values of redox-linked acid dissociations (deduced from changes in slope of the E(m) versus pH profiles) has also been determined for both E. coli thioredoxin and C. reinhardtii thioredoxin h.
Setterdahl
Aaron T
AT
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79401-1061, USA.
Chivers
Peter T
PT
Hirasawa
Masakazu
M
Lemaire
Stéphane D
SD
Keryer
Eliane
E
Miginiac-Maslow
Myroslawa
M
Kim
Sung-Kun
SK
Mason
Jeremy
J
Jacquot
Jean-Pierre
JP
Longbine
Christopher C
CC
de Lamotte-Guery
Frédéric
F
Knaff
David B
DB
D000409:Alanine / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D016825:Chlamydomonas reinhardtii / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004579:Electron Transport / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011522:Protons; D054480:Thioredoxin h; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics; D014364:Tryptophan / Q000235:genetics
D004220:Disulfides; D029968:Escherichia coli Proteins; D011522:Protons; D054480:Thioredoxin h; D001224:Aspartic Acid; D013879:Thioredoxins; D014364:Tryptophan; D003545:Cysteine; D000409:Alanine