Conformation state of mitochondrial complex I
Hypoxia is characterized by a lack of oxygen that is sufficient to cause a cellular response of the tissue. Many factors may cause hypoxia, including pathology, trauma, and it can also occur during surgery. Hypoxia is followed by restoration of the oxygen supply, or reoxygenation, which injures cells and causes potentially irreversible damage. Mitochondria are responsible for such reoxygenation damage during reperfusion when detrimental oxygen and nitrogen reactive species are formed. Mitochondrial Complex I is the main consumer of the NADH generated in the Krebs cycle, as well as a key component of the respiratory chain, contributing to the formation of potential across the membrane and consequently to ATP-synthesis. Complex I is also known to undergo reversible conformational changes in situations where its turnover is limited. As initially proposed in Andrei Vinogradov’s lab [Kotlyar&Vinogradov, 1990]; see also [Vinogradov, 1998] for a review): if the enzyme is idle at physiological temperatures (30°C), the mammalian enzyme is rapidly converted into the de-active, dormant form (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 (A-form) and can catalyze the physiological NADH:ubiquinone reaction at a much higher rate (k~104 min-1) (see Drose et al., 2016 for a review). A critical cysteine-39 of the mitochondrially-encoded ND3 subunit has been shown to become exposed on the surface of the D-form and can be specifically labeled by fluorescein-NEM (video and figure below) [Galkin et al., 2008]. This cysteine residue is not accessible to any covalent modification in the A-form and is responsible for the sensitivity of the D-form to NO-metabolites in isolated mitochondrial membranes and in cultured cells [Galkin and Moncada 2007; Galkin et al., 2009].
We demonstrated accumulation of the D-form of Complex I in cells after prolonged hypoxia or metabolic hypoxia when available oxygen cannot be used due to the increase in NO [Galkin et al., 2009]. In such a situation, cytochrome c oxidase activity is slowed down and the respiratory chain is reduced. Due to the absence of electron acceptor ubiquinone, which is in its ubiquinol form, turnover of the enzyme is decreased and Complex I undergoes de-activation. The details of that process are currently under investigation.
Recently we also showed the presence of an A/D transition in the neonatal brain [Stepanova et al., 2017]. Mitochondrially targeted nitrosothiol MitoSNO specifically modifies ND3 Cys-39 and results in attenuation of ischemia/reperfusion injury in the neonatal brain [Kim et al., 2018].
Our finding on the difference between the A and the D forms [Galkin et al., 2009; Babot et al., 2014; Ciano et al . 2013] were confirmed after the structure of both forms was published by the Judi Hirst group [Agip et al., 2018] (PDB structure of the mouse deactive and active enzyme are available now). The structural and functional studies of the A/D transition were initiated in the ’90s [Kotlyar&Vinogradov, 1990; Maklashina et al., 1994], and continue to be under investigation in several laboratories worldwide.
- Kim M, Stepanova A, Niatsetskaya Z, Sosunov S, Arndt S, Murphy MP, Galkin A, Ten V (2018) Attenuation of oxidative damage by targeting mitochondrial complex I in neonatal hypoxic-ischemic brain injury. Free Radic Biol Med. 124, 517-524. PMID:30037775, PDF
- Stepanova A, Konrad C, Guerrero-Castillo S, Manfredi G, Vannucci S, Arnold S, Galkin A. (2018) Deactivation of mitochondrial complex I after hypoxia-ischemia in the immature brain. J.Cerebral Blood Flow and Metabolism. 271678X18770331. PMID:29629602, PDF
- Galkin A., Moncada S. (2017) Modulation of the conformational state of mitochondrial complex I as a target for therapeutic intervention. Interface Focus, 7:20160104. PMID:28382200, PDF
- Dröse, S., Stepanova A., Galkin A. (2016) Ischemic A/D transition of mitochondrial complex I and its role in ROS generation. Biochim. Biophys. Acta. 1857, 946-957
- Babot M., Labarbuta P., Birch A., Kew S., Fuszard M., Botting C., Heide H., Wittig I., Galkin A. (2014) ND3, ND1 and 39 kDa subunits are more exposed in the de-active form of bovine mitochondrial complex I. Biochim. Biophys. Acta. 1837:929-939. PMID:24560811, PDF
- Gorenkova N., Robinson E., Grieve D., Galkin A. (2013) Conformational change of mitochondrial complex I increases ROS sensitivity during ischaemia. Antioxid Redox Signal. 19, 1459-68. PMID:23419200, PDF
- Galkin A., Abramov A., Frakich N., Duchen M., Moncada S. (2009) Lack of oxygen deactivates mitochondrial complex I: Implications for ischemic injury? J.Biol.Chem., 284: 36055-36061. PMID:19861410, PDF
- Galkin A., Meyer B., Wittig I., Karas M., Schägger H., Vinogradov A., Brandt U. (2008) Identification of the mitochondrial complex I ND3 subunit as a structural component involved in the active/de-active enzyme transition. J.Biol.Chem., 283: 20907-20913. PMID:18502755, PDF
- Galkin, A. and Moncada, S. (2007 )Nitrosation of mitochondrial complex I. J.Biol.Chem., 282: 37448-37453. PMID:17956863, PDF,