Proton pumping by an inactive structural variant of cytochrome c oxidase

The aa(3)-type cytochrome c oxidases (CytcOs) 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 CytcO, 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 CytcO 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 is 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. similar to 200 mu s and similar to 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. (C) 2014 Elsevier Inc. All rights reserved.