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

---

Ilya P. Oleynikov^1,2, Natalia V. Azarkina^2,a*, Tatiana V. Vygodina², and Alexander A. Konstantinov^2,#

¹Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119992 Moscow, Russia

²Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia

^* To whom correspondence should be addressed.

^# Deceased.

Received September 15, 2020; Revised October 30, 2020; Accepted October 30, 2020

---

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_i = 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]_1/2 = 0.28 mM, presumably associated with heme a₃. When CcO interacts with excess of H₂O₂ TX affects equilibrium of the oxygen intermediates of the catalytic center accelerating the F_I-607 → F_II-580 transition, inhibits generation of O₂^·— 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_II-580 to the free oxidized state during the catalytic cycle. TX suppresses intraprotein electron transfer between hemes a and a₃ 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.

---

KEY WORDS: cytochrome c oxidase, Triton X-100, regulation, K-cnannel, amphipathic ligands

DOI: 10.1134/S0006297921010053

---