Mechanism of rotenone binding to respiratory complex I depends on ligand flexibility

Abstract

Respiratory complex I is a major cellular energy transducer located in the inner mitochondrial membrane. Its inhibition by rotenone, a natural isoflavonoid, has been used for centuries by indigenous peoples to aid in fishing and, more recently, as a broad-spectrum pesticide or even a possible anticancer therapeutic. Unraveling the molecular mechanism of rotenone action will help to design tuned derivatives and to understand the still mysterious catalytic mechanism of complex I. Although composed of five fused rings, rotenone is a flexible molecule and populates two conformers, bent and straight. Here, a rotenone derivative locked in the straight form was synthesized and found to inhibit complex I with 600-fold less potency than natural rotenone. Large-scale molecular dynamics and free energy simulations of the pathway for ligand binding to complex I show that rotenone is more stable in the bent conformer, either free in the membrane or bound to the redox active site in the substrate-binding Q-channel. However, the straight conformer is necessary for passage from the membrane through the narrow entrance of the channel. The less potent inhibition of the synthesized derivative is therefore due to its lack of internal flexibility, and interconversion between bent and straight forms is required to enable efficient kinetics and high stability for rotenone binding. The ligand also induces reconfiguration of protein loops and side-chains inside the Q-channel similar to structural changes that occur in the open to closed conformational transition of complex I. Detailed understanding of ligand flexibility and interactions that determine rotenone binding may now be exploited to tune the properties of synthetic derivatives for specific applications.

H2S Protects from Rotenone-Induced Ferroptosis by Stabilizing Fe-S Clusters in Rat Cardiac Cells

“…In addition, the mRNA levels of mitoferrin 1 , mitoferrin 2 , and TFR were not affected by either rotenone and/or NaHS. Mitochondrial complex I activity has the largest number of iron-sulfur (Fe-S) cluster-containing proteins in the electron transport chain, which is known to be inhibited by rotenone [ 26 ]. ACO contains a [4Fe-4S]-cluster and is an essential enzyme in the citric acid cycle [ 7 ].…”

“…Higher levels of labile iron would induce oxidative stress, leading to mitochondrial damage, lipid peroxidation, and eventually ferroptosis [ 23 , 24 ]. Rotenone, a broad-spectrum pesticide, is a second-class hazardous agent for humans and is often used by researchers to mimic ischemic cell damage [ 25 , 26 , 27 ]. Many studies have shown that rotenone inhibits complex I in the mitochondrial ETC, leading to the disruption of ATP generation and initiation of cell death [ 25 , 28 , 29 ].…”

LRRK2 kinase inhibition protects against Parkinson’s disease-associated environmental toxicants

“…Likewise, WT cells were no longer protected from 1µM rotenone by MLi2. As 1 µM rotenone is likely approaching near complete complex I inhibition 67 , we suspect that overt toxicity overwhelms the protective effect of LRRK2-mediated effects such as improved mitochondrial clearance. However, as most exposures associated with PD likely happen at low doses over chronic periods 1,4,7,8,64,68,69 , LRRK2 could be involved in crosstalk with pathways triggered in parallel by environmental risk factors 26 .…”

Alternative Oxidase: From Molecule and Function to Future Inhibitors

“…It can be observed that these four binding sites share some common amino acid residues, which are conserved in various species ( Figure 4 A, blue dotted box) and likely to be conserved binding sites for TAO inhibitors ( Figure 7 K). The binding mechanism of rotenone to respiratory complex I is contingent upon the flexibility of the ligand; 176 it is also worthy to investigate the correlation between mechanism of this site and the flexibility of its ligands.…”