EFFECT OF CROSSLINKING ON MITOCHONDRIAL CYTOCHROME c OXIDASE

Purified and reconstituted cytpchrome ~oxidase and mitochondria were crosslinked with biimidates in the presence and absence of cytochrome c. These experiments indicate that oxidase subunit interactions are required for activity and that cytochrome~ mobility may be required for electron transport activity. Biimidate treatment of purified and reconstituted oxidase crosslinks all of the oxidase protomers except subunit I when ?20% of the free amines are modified and inhibits steady state oxidase activity. Transient kinetics of ferrocytochrome ~oxidation and ferricytochrome ~reduction indicates inhibition of electron transfer from heme ~to heme 3 . Crosslinking xidase molecules to form large aggregates displaying rotational correlation times >l ms does not affect oxidase activity. Crosslinking of mitochondria covalently binds the bc1 and 3 complexes to cytochrome ~' and inhibits steady-state oxidase activity considerably more than in the case of the purified oxidase. Addition of cytochrome~ to the purified oxidase or to ~-depleted mitoplasts increases inhibition slightly. Cytochrome~ oligomers act as competitive inhibitors of native ~· however, crosslinking of cytochrome~ to ~-depleted mitoplasts or purified oxidase (with dimethyl suberimidate or hetrobifunctional crosslinking reagents) results in a catalytically inactive complex.