Proton pumping by an inactive structural variant of cytochrome c oxidase.

Abstract The aa 3 -type cytochrome c oxidases (Cyt c Os) from e.g. Rhodobacter sphaeroides and Paracoccus denitrificans harbor two proton-transfer pathways. The K pathway is used for proton uptake upon reduction of the Cyt c O, while the D pathway is used after binding of O 2 to the catalytic site. The aim of the present study was to determine whether or not Cyt c O in which the K pathway is blocked (by e.g. the Lys362Met replacement) is capable of pumping protons. The process can not be studied using conventional assays because the O 2 -reduction activity is too low when the K pathway is blocked. Consequently, proton pumping with a blocked K pathway has not been demonstrated directly. Here, the Lys362Met and Ser299Glu structural variants were reconstituted in liposomes and allowed to (slowly) become completely reduced. Then, the reaction with O 2 was studied with μs time resolution after flash photolysis of a blocking CO ligand bound to heme a 3 . The data show that with both the inactive Lys362Met and partly active Ser299Glu variants proton release occurred with the same time constants as with the wild-type oxidase, i.e. ~ 200 μs and ~ 3 ms, corresponding in time to formation of the ferryl and oxidized states, respectively. Thus, the data show that the K pathway is not required for proton pumping, suggesting that D and K pathways operate independently of each other after binding of O 2 to the catalytic site.