Mechanism of Inhibition of Cytochrome c Oxidase by Triton X-100.

It is known that Triton X-100 (TX) reversibly inhibits activity of cytochrome c oxidase (CcO). The mechanism of inhibition is analyzed in this work. The action of TX is not directed to the reaction of CcO with cytochrome c, does not cause transition of the enzyme to the "slow" form, and is not associated with monomerization of the enzyme complex. TX completely suppresses oxygen reduction by CcO, but inhibition is prevented and partially reversed by dodecyl-β-D-maltoside (DDM), a detergent used to maintain CcO in solution. A 1/1 stoichiometry competition is shown between DDM and TX for binding to CcO, with K = 0.3 mM and affinity of DDM for the enzyme of 1.2 mM. TX interaction with the oxidized enzyme induces spectral response with maximum at 421 nm and [TX] = 0.28 mM, presumably associated with heme a. When CcO interacts with excess of H2O2 TX affects equilibrium of the oxygen intermediates of the catalytic center accelerating the F-607 → F-580 transition, inhibits generation of O by the enzyme, and, to a lesser extent, suppresses the catalase partial activity. The observed effects can be explained by inhibition of the conversion of the intermediate F-580 to the free oxidized state during the catalytic cycle. TX suppresses intraprotein electron transfer between hemes a and a during enzyme turnover. Partial peroxidase activity of CcO remains relatively resistant to TX under conditions that block oxidase reaction effectively. These features indicate an impairment of the K proton channel conductivity. We suggest that TX interacts with CcO at the Bile Acid Binding Site (BABS) that is located on the subunit I at the K-channel mouth and contacts with amphipathic regulators of CcO [Buhrow et al. (2013) Biochemistry, 52, 6995-7006]. Apparently, TX mimics the physiological ligand of BABS, whereas the DDM molecule mimics an endogenous phospholipid bound at the edge of BABS that controls effective affinity for the ligand.