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 denitrificans interacts with the formyl group of heme a. Mutation of this arginine to methionine (R54M) dramatically changes the spectral properties of heme a and 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 of 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 electron transfer is impaired thermodynamically due to the low redox potential of heme a but that it still takes place from Cu(A) 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 efficiency is unaffected at 1 H(+)/e(-) in the mutant enzyme, although turnover is strongly inhibited. Time-resolved electrometry shows that when the fully reduced enzyme reacts with O(2), the fast phase of membrane potential generation during the P(R )()--> F transition is unaffected by the mutation, whereas the slow phase (F --> O transition) is strongly decelerated. In the 3e(-)-reduced mutant enzyme heme a remains oxidized due to its lowered midpoint potential, whereas Cu(A) and the binuclear site are reduced. In this case the reaction with O(2) proceeds via the P(M) state because transfer of the electron from Cu(A) to the binuclear site is delayed. The single phase of membrane potential generation in the 3e(-)-reduced mutant enzyme, which thus corresponds to the P(M)--> F transition, is decelerated, but its amplitude is comparable to that of the P(R)--> F transition. From this we conclude that the completely (4e(-)) reduced enzyme is fully capable of proton translocation.
Jasaitis
A
A
Helsinki Bioenergetics Group, Department of Medical Chemistry, Institute of Biomedical Sciences and Biocentrum Helsinki, University of Helsinki, Finland.
Backgren
C
C
Morgan
J E
JE
Puustinen
A
A
Verkhovsky
M I
MI
Wikström
M
M
D001120:Arginine / Q000235:genetics*; D001692:Biological Transport; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006418:Heme / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials; D008715:Methionine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010231:Paracoccus denitrificans / Q000201:enzymology* / Q000235:genetics; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D010782:Photolysis; D011522:Protons; D013053:Spectrophotometry
D010743:Phospholipids; D011522:Protons; C027728:heme a; D006418:Heme; D003300:Copper; D001120:Arginine; D008715:Methionine; D003576:Electron Transport Complex IV; D010100:Oxygen