Structural basis of mammalian complex IV inhibition by steroids

Abstract

The mitochondrial electron transport chain maintains the proton motive force that powers adenosine triphosphate (ATP) synthesis. The energy for this process comes from oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate, with the electrons from this oxidation passed via intermediate carriers to oxygen. Complex IV (CIV), the terminal oxidase, transfers electrons from the intermediate electron carrier cytochrome c to oxygen, contributing to the proton motive force in the process. Within CIV, protons move through the K and D pathways during turnover. The former is responsible for transferring two protons to the enzyme’s catalytic site upon its reduction, where they eventually combine with oxygen and electrons to form water. CIV is the main site for respiratory regulation, and although previous studies showed that steroid binding can regulate CIV activity, little is known about how this regulation occurs. Here, we characterize the interaction between CIV and steroids using a combination of kinetic experiments, structure determination, and molecular simulations. We show that molecules with a sterol moiety, such as glyco-diosgenin and cholesteryl hemisuccinate, reversibly inhibit CIV. Flash photolysis experiments probing the rapid equilibration of electrons within CIV demonstrate that binding of these molecules inhibits proton uptake through the K pathway. Single particle cryogenic electron microscopy (cryo-EM) of CIV with glyco-diosgenin reveals a previously undescribed steroid binding site adjacent to the K pathway, and molecular simulations suggest that the steroid binding modulates the conformational dynamics of key residues and proton transfer kinetics within this pathway. The binding pose of the sterol group sheds light on possible structural gating mechanisms in the CIV catalytic cycle.

Long-range charge transfer mechanism of the III2IV2 mycobacterial supercomplex

“…On the Complex IV side of the SC, our combined MD and cryo-EM data show hydrogen-bonded water arrays along the D- and K-channels that support the proton uptake from the N-side of the membrane to Glu266 (via the D-channel) or via Lys340 to Tyr268 of the BNC (via the K-channel) (Fig. 4d–f ) 38 , 39 (cf. also refs.…”

“…See ref. 39 for further details of the simulation setup of the bovine Complex IV simulations. Survival probabilities of water molecules in the D- and K-channel were calculated using a spherical volume with a 5 Å radius around residues Thr125 and Thr337.…”

The Catalytic Reaction of Cytochrome c Oxidase Probed by In Situ Gas Titrations and FTIR Difference Spectroscopy

“…The effects of cavity hydration and local electric fields have been proposed to influence the trajectories of protons leading towards the PLS [46,57]. There have been proposals for structural gating mechanism in mitochondrial CcO [58] and supercomplexes have been identified [59] further reinforcing the idea that membrane dynamics and non-diffusion -riven reaction are key to the function of CcOs.…”

Phytosterol organic acid esters: Characterization, anti-inflammatory properties and a delivery strategy to improve mitochondrial function

“…However, the limited water solubility of phytosterols such as STI restricts their application ( He et al, 2018 ; Sakamoto et al, 2013 ; Witkowska et al, 2022 ). Still, as a cholesterol analog, STI acts as an effective metabolic regulator ( Di Trani et al, 2022 ).…”