Structural basis of mammalian Complex IV inhibition by steroids

Abstract

The mitochondrial electron transport chain maintains the proton motive force that powers ATP synthesis. The energy for this process comes from oxidation of 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 enzymes catalytic site upon reduction of the site, 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 high-speed equilibration of electrons within CIV demonstrate that binding of these molecules inhibits proton uptake through the K-pathway. Single particle 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. SIGNIFICANCE STATEMENTMammalian complex IV (CIV), the final complex of the mitochondrial electron transport chain, uses electrons from cytochrome c to reduce oxygen to water, driving aerobic life. Although CIV functions as the main site for respiratory regulation, there is little structural or biochemical information on how this regulation occurs. Previous studies provided evidence of CIV regulation by steroids, but the steroid binding site and regulatory mechanism remain unclear. Using single particle cryogenic electron microscopy, we discover the binding site of the steroid-derived detergent, glyco-diosgenin. Results from flash photolysis kinetic experiments with CIV in the presence of glyco-diosgenin and cholesterol hemisuccinate are combined with cryo-EM and molecular simulations to elucidate how steroid binding limits proton uptake by the complex.