Electron and proton transfer in the arginine-54-methionine mutant of cytochrome c oxidase from Paracoccus denitrificans

Arginine 54 in subunit I of cytochrome c oxidase from Paracoccus denitrific ans interacts with the formyl group of heme a. Mutation of this arginine to methionine (R54M) dramatically changes the spectral properties of heme a a nd lowers its midpoint redox potential [Kannt et al, (1999) J. Biol. Chem. 274, 37974-37981; Lee et al, (2000) Biochemistry 39, 2989-2996; Riistama et al. (2000) Biochim. Biophys. Acta 1456, 1-4]. During anaerobic reduction o f the mutant enzyme, a small fraction of heme a is reduced first along with heme a(3), while most of heme a is reduced later. This suggests that elect ron transfer is impaired thermodynamically due to the low redox potential o f heme a but that it still takes place from CUA via heme a to the binuclear site as in wild-type enzyme, with no detectable bypass from Cu-A directly to the binuclear site. Consistent with this, the proton translocation effic iency is unaffected at 1 H+/e(-) in the mutant enzyme, although turnover is strongly inhibited. Time-resolved electrometry shows that when the fully r educed enzyme reacts with O-2, the fast phase of membrane potential generat ion during the PR --> F transition is unaffected by the mutation, whereas t he slow phase (F --> O transition) is strongly decelerated. In the 3e(-)-re duced mutant enzyme heme a remains oxidized due to its lowered midpoint pot ential, whereas Cu-A and the binuclear site are reduced. In this case the r eaction with O-2 proceeds via the P-M state because transfer of the electro n from CuA to the binuclear site is delayed. The single phase of membrane p otential generation in the 3e--reduced mutant enzyme, which thus correspond s to the P-M --> F transition, is decelerated, but its amplitude is compara ble to that of the P-R --> F transition.