ADP- and oligomycin-sensitive redox behavior of F0 b thiol in ATPsynthase depends on neighbored primary structure: investigations using 14-C-labeled alpha lipoic acid.
Purified ATPsynthase of bovine heart mitochondria has been analyzed for its mobility and reactivity of oligomycin-sensitive sulfhydryl regions in presence of the substrate ADP and oligomycin. Labeling of thiol groups at the hydrophobic F_0 region of the ATPsynthase was increased in the enzyme initially treated with SDS, N-ethylmaleimide and dithiothreitol (modified enzyme). After dialysis or gel permeation the ATPsynthase was treated with [14C] alpha lipoic acid at a molar ratio of 35-85/1 (lipoic acid/ATPsynthase) corresponding to 4-8.6 nmol/mg protein. Under these conditions, ATPase activity of the native enzyme was significantly decreased. After preincubation with ADP, PAGE of the native, [14C] labeled enzyme revealed an increase of radioactivity at a region of 25 kDa deduced to Cys 197 of subunit b. In the modified enzyme the increase in radioactivity was found at 10 kDa. In this context, the sequence Lys-Cys-Ile around Cys 197 of subunit b suggests excessive reactivity of this thiol, as well as ready reversibility by -SH-S-S- interchange. Therefore, previously observed reaction by thiol reagents and antioxidants from outside the mitochondrion can be interpreted with Cys 197 of F0 b. It accounts for sulfhydryl unmasked by binding of ADP at F1.
Dünschede
Fritz
F
Allgemein- und Abdominalchirurgie, Universitätsklinik Mainz, Germany.
Zwicker
Klaus
K
Ackermann
Hanns
H
Zimmer
Guido
G
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D002250:Carbon Radioisotopes; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D008929:Mitochondria, Heart / Q000201:enzymology; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D009840:Oligomycins / Q000494:pharmacology*; D010084:Oxidation-Reduction / Q000187:drug effects; D021122:Protein Subunits / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013113:Spin Labels; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000737:chemistry / Q000378:metabolism; D008063:Thioctic Acid / Q000737:chemistry* / Q000378:metabolism
D000975:Antioxidants; D002250:Carbon Radioisotopes; D009840:Oligomycins; D021122:Protein Subunits; D013113:Spin Labels; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D000244:Adenosine Diphosphate; D008063:Thioctic Acid; D025261:Mitochondrial Proton-Translocating ATPases