Комплекс I дыхательной цепи: структура, функция и ингибиторы.

Protein complex involved in cellular respiration

Respiratory complex I, (also known as NADH:ubiquinone oxidoreductase, Type I NADH dehydrogenase and mitochondrial complex I) is the first large protein complex of the respiratory chains of many organisms from bacteria to humans. It catalyzes the transfer of electrons from NADH to coenzyme Q10 (CoQ10) and translocates protons across the inner mitochondrial membrane in eukaryotes or the plasma membrane of bacteria. This enzyme is essential for the normal functioning of cells, and mutations in its subunits lead to a wide range of inherited neuromuscular and metabolic disorders. Defects in this enzyme are responsible for the development of several pathological processes such as ischemia/reperfusion damage (stroke and cardiac infarction), Parkinson's disease and others.

All redox reactions take place in the hydrophilic domain of complex I. NADH initially binds to complex I, and transfers two electrons to the flavin mononucleotide (FMN) prosthetic group of the enzyme, creating FMNH2. The electron acceptor – the isoalloxazine ring – of FMN is identical to that of FAD. The electrons are then transferred through the FMN via a series of iron sulfur (Fe S) clusters, Ubiquinone (CoQ) accepts two electrons to be reduced to ubiquinol (CoQH2). The high reduction potential of the N2 cluster and the relative proximity of the other clusters in the chain enable efficient electron transfer over long distance in the protein (with transfer rates from NADH to N2 iron sulfur cluster of about 100 μs). The equilibrium dynamics of Complex I are primarily driven by the quinone redox cycle. In conditions of high proton motive force (and accordingly, a ubiquinol concentrated pool), the enzyme runs in the reverse direction. Ubiquinol is oxidized to ubiquinone, and the resulting released protons reduce the proton motive force.

Bullatacin (an acetogenin found in Asimina triloba fruit) is the most potent known inhibitor of NADH dehydrogenase (ubiquinone) (=1.2 nM, stronger than rotenone). The best known inhibitor of complex I is rotenone (commonly used as an organic pesticide). Rotenone and rotenoids are isoflavonoids occurring in several genera of tropical plants such as Antonia (Loganiaceae), Derris and Lonchocarpus (Faboideae, Fabaceae). There have been reports of the indigenous people of French Guiana using rotenone containing plants to fish due to its ichthyotoxic effect as early as the 17th century. Rotenone binds to the ubiquinone binding site of complex I as well as piericidin A, another potent inhibitor with a close structural homologue to ubiquinone. Acetogenins from Annonaceae are even more potent inhibitors of complex I. They cross link to the ND2 subunit, which suggests that ND2 is essential for quinone binding. Rolliniastatin 2, an acetogenin, is the first complex I inhibitor found that does not share the same binding site as rotenone. Despite more than 50 years of study of complex I, no inhibitors blocking the electron flow inside the enzyme have been found. Hydrophobic inhibitors like rotenone or piericidin most likely disrupt the electron transfer between the terminal FeS cluster N2 and ubiquinone. It has been shown that long term systemic inhibition of complex I by rotenone can induce selective degeneration of dopaminergic neurons. Complex I is also blocked by adenosine diphosphate ribose – a reversible competitive inhibitor of NADH oxidation – by binding to the enzyme at the nucleotide binding site. Both hydrophilic NADH and hydrophobic ubiquinone analogs act at the beginning and the end of the internal electron transport pathway, respectively. The antidiabetic drug Metformin has been shown to induce a mild and transient inhibition of the mitochondrial respiratory chain complex I, and this inhibition appears to play a key role in its mechanism of action. Inhibition of complex I has been implicated in hepatotoxicity associated with a variety of drugs, for instance flutamide and nefazodone. Further, complex I inhibition was shown to trigger NAD+ independent glucose catabolism.

The catalytic properties of eukaryotic complex I are not simple. Two catalytically and structurally distinct forms exist in any given preparation of the enzyme: one is the fully competent, so called “active” A form and the other is the catalytically silent, dormant, “inactive”, D form. After exposure of idle enzyme to elevated, but physiological temperatures (>30 °C) in the absence of substrate, the enzyme converts to the D form. This form is catalytically incompetent but can be activated by the slow reaction (k~4 min−1) of NADH oxidation with subsequent ubiquinone reduction. After one or several turnovers the enzyme becomes active and can catalyse physiological NADH:ubiquinone reaction at a much higher rate (k~104 min−1). In the presence of divalent cations (Mg2+, Ca2+), or at alkaline pH the activation takes much longer. The high activation energy (270 kJ/mol) of the deactivation process indicates the occurrence of major conformational changes in the organisation of the complex I. However, until now, the only conformational difference observed between these two forms is the number of cysteine residues exposed at the surface of the enzyme. Treatment of the D form of complex I with the sulfhydryl reagents N Ethylmaleimide or DTNB irreversibly blocks critical cysteine residues, abolishing the ability of the enzyme to respond to activation, thus inactivating it irreversibly. The A form of complex I is insensitive to sulfhydryl reagents. It was found that these conformational changes may have a very important physiological significance. The inactive, but not the active form of complex I was susceptible to inhibition by nitrosothiols and peroxynitrite. It is likely that transition from the active to the inactive form of complex I takes place during pathological conditions when the turnover of the enzyme is limited at physiological temperatures, such as during hypoxia, ischemia or when the tissue nitric oxide:oxygen ratio increases (i. e. metabolic hypoxia).

Recent investigations suggest that complex I is a potent source of reactive oxygen species. Complex I can produce superoxide (as well as hydrogen peroxide), through at least two different pathways. During forward electron transfer, only very small amounts of superoxide are produced (probably less than 0.1% of the overall electron flow). During reverse electron transfer, complex I might be the most important site of superoxide production within mitochondria, with around 3 4% of electrons being diverted to superoxide formation. Reverse electron transfer, the process by which electrons from the reduced ubiquinol pool (supplied by succinate dehydrogenase, glycerol 3 phosphate dehydrogenase, electron transferring flavoprotein or dihydroorotate dehydrogenase in mammalian mitochondria) pass through complex I to reduce NAD+ to NADH, driven by the inner mitochondrial membrane potential electric potential. Although it is not precisely known under what pathological conditions reverse electron transfer would occur in vivo, in vitro experiments indicate that this process can be a very potent source of superoxide when succinate concentrations are high and oxaloacetate or malate concentrations are low. This can take place during tissue ischaemia, when oxygen delivery is blocked. Superoxide is a reactive oxygen species that contributes to cellular oxidative stress and is linked to neuromuscular diseases and aging. NADH dehydrogenase produces superoxide by transferring one electron from FMNH2 (or semireduced flavin) to oxygen (O2). The radical flavin leftover is unstable, and transfers the remaining electron to the iron sulfur centers. It is the ratio of NADH to NAD+ that determines the rate of superoxide formation.

Mutations in the subunits of complex I can cause mitochondrial diseases, including Leigh syndrome. Point mutations in various complex I subunits derived from mitochondrial DNA (mtDNA) can also result in Leber's Hereditary Optic Neuropathy. There is some evidence that complex I defects may play a role in the etiology of Parkinson's disease, perhaps because of reactive oxygen species (complex I can, like complex III, leak electrons to oxygen, forming highly toxic superoxide). Although the exact etiology of Parkinson's disease is unclear, it is likely that mitochondrial dysfunction, along with proteasome inhibition and environmental toxins, may play a large role. In fact, the inhibition of complex I has been shown to cause the production of peroxides and a decrease in proteasome activity, which may lead to Parkinson's disease. Additionally, Esteves et al. (2010) found that cell lines with Parkinson's disease show increased proton leakage in complex I, which causes decreased maximum respiratory capacity. Brain ischemia/reperfusion injury is mediated via complex I impairment. Recently it was found that oxygen deprivation leads to conditions in which mitochondrial complex I lose its natural cofactor, flavin mononucleotide (FMN) and become inactive. When oxygen is present the enzyme catalyzes a physiological reaction of NADH oxidation by ubiquinone, supplying electrons downstream of the respiratory chain (complexes III and IV). Ischemia leads to dramatic increase of succinate level. In the presence of succinate mitochondria catalyze reverse electron transfer so that fraction of electrons from succinate is directed upstream to FMN of complex I. Reverse electron transfer results in a reduction of complex I FMN, Similarly, Moran et al. (2010) found that patients with severe complex I deficiency showed decreased oxygen consumption rates and slower growth rates. However, they found that mutations in different genes in complex I lead to different phenotypes, thereby explaining the variations of pathophysiological manifestations of complex I deficiency. Exposure to pesticides can also inhibit complex I and cause disease symptoms. For example, chronic exposure to low levels of dichlorvos, an organophosphate used as a pesticide, has been shown to cause liver dysfunction. This occurs because dichlorvos alters complex I and II activity levels, which leads to decreased mitochondrial electron transfer activities and decreased ATP synthesis.

A proton pumping, ubiquinone using NADH dehydrogenase complex, homologous to complex I, is found in the chloroplast genomes of most land plants under the name ndh. This complex is inherited from the original symbiosis from cyanobacteria, but has been lost in most eukaryotic algae, some gymnosperms (Pinus and gnetophytes), and some very young lineages of angiosperms. The purpose of this complex is originally cryptic as chloroplasts do not participate in respiration, but now it is known that ndh serves to maintain photosynthesis in stressful situations. This makes it at least partially dispensable in favorable conditions. It is evident that angiosperm lineages without ndh do not last long from their young ages, but how gymnosperms survive on land without ndh for so long is unknown.