Effect of papain digestion on polypeptide subunits and electron-transfer pathways in mitochondrial b-c1 complex.
Papain digestion of subunits of mitochondrial b-c1 complex (ubiquinol-cytochrome-c reductase) isolated from bovine heart and its impact on redox and proton-motive activity of the whole complex were investigated. A 5-min incubation of the oxidized enzyme with papain resulted in digestion of core protein II and the 14-kDa subunit, and limited digestion of the iron-sulfur protein. This was accompanied by a small inhibition of the rate of electron flow and a marked inhibition of proton translocation with decrease of the H+/e- ratio for proton pumping. When papain treatment was performed on the b-c1 complex pre-reduced with ascorbate, partial proteolysis of the iron-sulfur protein and the 14-kDa subunit was greatly accelerated and the electron transfer activity was more markedly inhibited. In all the conditions tested, digestion of the Rieske iron-sulfur protein paralleled the inhibition of reductase activity. Under ascorbate-reduced conditions, papain digestion of the complex gave rise to an alteration of the EPR line shape of the iron-sulfur cluster, namely a broadening and shift of the gx negative peak and destabilization of the protein-bound antimycin-sensitive semiquinone. The latter paralleled the decrease in electron transfer activity and inhibition of antimycin-sensitive cytochrome-b reduction. The results obtained indicate the following. 1. Core protein II and the 14-kDa protein may contribute to the proton-conducting pathway(s) from the matrix aqueous phase to the primary protolytic redox center (protein-bound semiquinone/quinone couple). 2. The iron-sulfur protein contributes, together with other protein(s) (the 14-kDa subunit), to the stabilization of the protein-bound antimycin-sensitive semiquinone species in a protein pocket in the complex. 3. Reduction of the high-potential redox centers induces a change in the quaternary structure of the complex which results in an enhanced surface exposure of segments of the 14-kDa protein and the iron-sulfur protein.
Lorusso
M
M
Institute of Medical Biochemistry and Chemistry, University of Bari, Italy.
Cocco
T
T
Boffoli
D
D
Gatti
D
D
Meinhardt
S
S
Ohnishi
T
T
Papa
S
S
D000818:Animals; D000968:Antimycin A / Q000031:analogs & derivatives / Q000494:pharmacology; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007506:Iron-Sulfur Proteins / Q000032:analysis; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D010084:Oxidation-Reduction; D010206:Papain; D010446:Peptide Fragments / Q000032:analysis*; D010455:Peptides / Q000032:analysis*; D011522:Protons; D013053:Spectrophotometry
D007506:Iron-Sulfur Proteins; D010446:Peptide Fragments; D010455:Peptides; D011522:Protons; C032456:antimycin; D000968:Antimycin A; D010206:Papain; D014450:Electron Transport Complex III