Effect of papain digestion on polypeptide subunits and electron‐transfer pathways in mitochondrial b‐c1 complex

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Abstract
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 antimyein‐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.