Binding of inhibitor, NADH-ubiquinone oxidoreductase (complex I)

Electron transport through the mitochondrial respiratory chain (oxidative phosphorylation) is mediated by five multimeric complexes (I–V) that are embedded in the mitochondrial inner membrane (Fig. 1). NADH-ubiquinone oxidoreductase is the Complex I (CI) of electron transport chain (ETC). It is a large assembly of proteins that spans the inner mitochondrial membrane. In mammals, it is composed of about 45-47 protein subunits (human 45) of which 7 are encoded by the mitochondrial genome (ND1, ND2, ND3, ND4, ND4L, ND5, and ND6) and the remainder by the nuclear genome (Greenamyre, 2001). CI oxidizes NADH elevating the NAD+/NADH ratio by transferring electrons via a flavin mononucleotide (FMN) cofactor and several iron-sulfur centers to ubiquinone (Friedrich et al., 1994) (Fig. 1). Binding of an inhibitor to CI inhibits the NADH–ubiquinone oxido-reductase activity, i.e. blocks the electron transfer. Recent studies suggest that a wide variety of CI inhibitors share a common binding domain at or close to the ubiquinone reduction site (Ino et al., 2003). Furthermore, the structural factors required for inhibitory actions have been characterized on the basis of structure-activity relationships (Miyoshi, 1998, Hideto, 1998). Based on molecular docking simulations, in silico models mimicking the binding of chemicals to the pocket of NADH ubiquinone oxidoreductase have been created according to the crystal structure of mitochondrial CI. To investigate the ability of chemicals to bind to the active pocket, around 100 individual docking simulations have been performed. These confirmed the possible site of interaction between the chemical and the pocket of CI. In particular, Miao YJ and coworkers recently investigated the IC50 values of 24 chemicals (annonaceous acetogenins) for inhibition of mitochondrial CI (Miao et al., 2014).

Based on their binding sites, CI inhibitors are classified as follows (Degli Esposti, 1998) (Fig. 2): (i) type A inhibitors are antagonists of fully oxidized ubiquinone binding; (ii) type B inhibitors displace the partially reduced ubisemiquinone intermediate; (iii) type C inhibitors are antagonists of the fully reduced ubiquinol product. The affinity of the different types of CI inhibitors to their diverse CI binding sites is described in the paragraph Evidence for Chemical Initiation of this Molecular Initiating Event (see below) in the context of a specific type of inhibitor.

Fig. 1. The electron transport chain in the mitochondrion. CI (NADH-coenzyme Q reductase or NADH dehydrogenase) accepts electrons from NADH and serves as the link between glycolysis, the citric acid cycle, fatty acid oxidation and the electron transport chain. Complex II also known as succinate-coenzyme Q reductase or succinate dehydrogenase, includes succinate dehydrogenase and serves as a direct link between the citric acid cycle and the electron transport chain. The coenzyme Q reductase or Complex III transfers the electrons from CoQH2 to reduce cytochrome c which is the substrate for Complex IV (cytochrome c reductase). Complex IV transfers the electrons from cytochrome c to reduce molecular oxygen into water. Finally, this gradient is used by the ATP synthase complex (Complex V) to make ATP via oxidative phosphorylation. mtDNA: mitochondrial DNA; nDNA: nuclear DNA.

Fig. 2. Schematic representation of CI and proposed inhibition binding sites by inhibitors of class A, B and C. Nicotinamide adenine dinucleotide (NADH, reduced and NAD, oxidized), flavin mononucleotide (FMN) and Ubiquinone (Q) (taken from Haefeli, 2012).

Two different types of approaches have been used. The first is to measure binding as such, and the corresponding assays are described below; the second is to infer binding indirectly from assays that quantify e.g. CI activity and to assume that the activity can only be altered upon binding. The second type of approach is dealt with in the chapter entitled KE1: Inhibition of NADH ubiquinone oxidoreductase (complex I). However, it has to be noted here that indirect assays can lead to wrong conclusions. For instance, some compounds may trigger oxidative stress without actually binding to CI. Such compounds, by triggering the generation of reactive oxygen species (ROS), may damage CI protein components, thus causing a reduction of CI activity.

Measurement of binding by quantitative autoradiography

To assess binding of an inhibitor at the rotenone binding site of CI in tissues (e.g. in the substantia nigra or in the striatum), the standard approach is to quantify the displacement of a radioactively labelled ligand of this binding site by the toxicant under evaluation. Most commonly, binding of [3H]-labeled dihydrorotenone (DHR) is measured and compared in control tissue and treated tissue. Binding of this rotenone-derivative is detected by autoradiography. Unselective binding is determined by measurement of [3H]-DHR binding in the presence of an excess of unlabeled rotenone. Since a rotenone-derivative is used for the assay, only CI inhibitors that bind to the rotenone-binding site in CI are detected. This was observed for e.g., meperdine, amobarbital, or MPP+. This method allows a spatial resolution of CI expression and the mapping of the binding of a competitive inhibitor on CI.

The method can be used for (a) in vitro measurements and for (b) ex vivo measurements:

a) In vitro measurements. Tissues are embedded in a matrix for cutting by a cryostat. The tissue slices are then mounted onto slides. For the binding experiment, they are incubated with the test compound in the presence of labeled [3H]-DHR. Then the tissue slices are washed and prepared for autoradiographic detection (Greenamyre et al. 1992; Higgins and Greenamyre, 1996). b) Ex vivo measurements. As rotenone can pass the blood brain barrier, the in vitro method was further extended for in vivo labeling of CI in the brains of living animals, and detection of binding after preparation of the tissue from such animals. Animals are exposed to test compounds and [3H]-DHR is applied intraventricularly for 2-6 h before the brain is dissected and arranged for the preparation of tissue slices (Talpade et al. 2000). In untreated animals, this method allows a precise spatial resolution of the expression pattern of CI. In animals with impaired CI activity, either as a result of CI deficiencies, or upon treatment with CI inhibitors, the assay allows an assessment of the degree of CI inhibition.

Complex I Enzyme Activity (Colorimetric)

The analysis of mitochondrial OXPHOS CI enzyme activity can be performed using human, rat, mouse and bovine cell and tissue extracts (abcam: http://www.abcam.com/complex-i-enzyme-activity-microplate-assay-kit-colorimetric-ab109721). Capture antibodies specific for CI subunits are pre-coated in the microplate wells. Samples are added to the microplate wells which have been pre-coated with a specific capture antibody. After the target has been immobilized in the well, CI activity is determined by following the oxidation of NADH to NAD+ and the simultaneous reduction of a dye which leads to increased absorbance at OD=450 nm. By analyzing the enzyme's activity in an isolated context, outside of the cell and free from any other variables, an accurate measurement of the enzyme's functional state can be evaluated.

CI has a highly conserved subunit composition across species, from lower organisms to mammals (Cardol, 2011). Fourteen subunits are considered to be the minimal structural requirement for physiological functionality of the enzyme. These units are well conserved among bacterial (E. coli), human (H. sapiens), and Bovine (B. taurus) (Vogel et al., 2007b; Ferguson, 1994). However, the complete structure of CI is reported to contain between 40 to 46 subunits and the number of subunits differs, depending on the species (Gabaldon 2005; Choi et al., 2008). In vertebrates CI consists of at least 46 subunits (Hassinen, 2007), particularly, in humans 45 subunits have been described (Vogel et al, 2007b). Moreover, enzymatic and immunochemical evidence indicate a high degree of similarity between mammalian and fungal counterparts (Lummen, 1998). Mammalian CI structure and activity have been characterized in detail (Vogel et al., 2007a; Vogel et al., 2007b), referring to different human organs including the brain. There is also a substantial amount of studies describing CI in human muscles, brain, liver, as well as bovine heart (Janssen et al., 2006; Mimaki et al. 2012) (Okun et al., 1999).

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