Detection of conformational changes in complex III of the respiratory chain by a maleimido spin label.
Changes in the conformation of Complex III (CoQH2-cytochrome c reductase) of the mitochondrial respiratory chain were detected upon oxidoreduction using the nitroxide spin label, 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl. EPR spectra of the spin label show a transition from a greater to a lesser degree of immobilization when the labeled enzyme, reduced either with ascorbate or sodium dithionite, is oxidized with potassium ferricyanide or ferricytochrome c. These observations are interpreted to indicate that Complex III is more compact in the reduced state at least in the locality of the spin label. An apparent increase in the concentration of total spins during oxidation of the complex suggests change in the interaction between the spin label and other paramagnetic centers and not an oxidation of spin label, itself, since reduced free spin label could not be reoxidized. Addition of antimycin A had no effect on the EPR spectrum of the spin-labeled enzyme, indicating that this inhibitor does not initiate a conformational change in the region of the spin label. Experiments in which N-ethyl-[2-3H] maleimide was bound to Complex III show that binding occurs primarily to a subunit with a molecular weight of 45,000. Although no qualitative differences were observed, it was found that less radioactivity appears in samples reduced with dithionite than in those reduced with ascorbate. This difference appears to be caused by decomposition products of dithionite.
D001205:Ascorbic Acid; D003497:Cyclic N-Oxides; D003574:Cytochrome c Group; D004227:Dithionite; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III; D005033:Ethylmaleimide; D005292:Ferricyanides; D008301:Maleimides; D008928:Mitochondria / Q000378:metabolism; D009097:Multienzyme Complexes; D009247:NADH, NADPH Oxidoreductases; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011808:Quinone Reductases; D013053:Spectrophotometry; D013113:Spin Labels
D003497:Cyclic N-Oxides; D003574:Cytochrome c Group; D005292:Ferricyanides; D008301:Maleimides; D009097:Multienzyme Complexes; D013113:Spin Labels; D004227:Dithionite; C024516:N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide; D009247:NADH, NADPH Oxidoreductases; D011808:Quinone Reductases; D014450:Electron Transport Complex III; D005033:Ethylmaleimide; D001205:Ascorbic Acid