Alexander Galkin



Bioenergetics, mitochondria, Respiratory chain, oxidative phosphorylation

Figure: Average human cell (top, middle) , mitochondria (top, right) and respiratory chain (bottom). I, Complex I or NADH: ubiquinone oxidoreductase; II, Complex II or succinate dehydrogenase; III, Complex III or bc1 complex or ubiquinol:cytochrome c oxidoreductase; IV, Complex IV or cytochrome c oxidase; Q/QH2 oxidised and reduced membrane ubiquinone, respectively; FMN, tightly bound flavin mononucleotide of Complex I; Succ, succinate; Fum, fumarate; O2 .-, superoxide anion; Qo.- and Qi.-, semiquinones of complex III; H+, protons translocated across the membrane; IMS, intermembrane space.

Respiratory chain oxidizes substrates and transfers electrons to oxygen. This process is coupled with translocation of protons (H+) from matrix side to IMS and, therefore, formation of proton-motive force. The flow of protons back to the matrix drives formation of ATP by Complex V or ATP synthase. Most of the energy in our body is generated by this mechanism which is  called oxidative phosphorylation.


MRC, NIH, grants, funding, Alexander Galkin

Mitochondrial complex I plays a critical role in regulating energy generation in cell and it is involved in a number of clinical conditions such as neurodegeneration (Parkinson’s disease, Leber’s optic neuropathy), muscular pathologies and the processes of ageing. At the same time, ischaemia/reperfusion injury mediated by mitochondria is a major cause of heart disease, as well as stroke; these two are the leading causes of death in the modern world, ranking before cancer and causing more than 30% of deaths. Our research is aimed at unraveling the role of mitochondrial complex I and other mitochondrial enzymes in such pathological conditions, translating our new findings to clinical medicine.


Mitochondrial ROS Production and Oxygen Level: Linear Dependence or Not?

Reactive oxygen species (ROS) are by-products of aerobic mitochondrial metabolism that are involved in both physiological cellular signaling pathways and pathophysiological processes of tissue damage including brain ischemia-reperfusion injury. The mitochondrial respiratory chain is considered a major source of ROS; however, there is little agreement on how ROS production depends on oxygen concentration ([O2]). We measured H2O2 release by intact mouse brain mitochondria during oxidation of different substrates at various oxygen concentrations. We found the highest rate of H2O2 release occurs under conditions of reverse electron transfer when mitochondria oxidize succinate or glycerol 3-phosphate and fraction of electrons (3-5%) goes upstream to Complex I.

For the first time, we determined that H2O2 release by respiring mitochondria depends linearly on the oxygen concentration in the physiological range (<200µM [O2]) during both reverse and forward electron transfer and with any substrate used (malate/pyruvate, succinate, succinate/glutamate or glycerol 3-phosphate). Our results suggest that ROS generation by brain mitochondria depends linearly on oxygen concentration obeying the law of mass action. We found that Complex III is not the major contributor to ROS generation under physiological conditions. H2O2 production by complex III is significant only in the presence of antimycin A, and in this case the oxygen dependence manifested mixed (linear and hyperbolic) kinetics. The hyperobolic component is contributed by Complex III (Qo site). Our results underscore the critical importance of reverse electron transfer in the brain, where a significant amount of succinate can be accumulated during ischemia and provide backflow of electrons upstream to complex I at the early stages of reperfusion. Our study [Stepanova et al., 2019] demonstrates a linear dependence of mitochondrial H2O2 release on oxygen concentration in the absence of inhibitors, indicating that ROS production in brain mitochondria under hypoxia is lower than in normoxia.

However, the question still persists - why does antimycin-induced production of ROS by complex III manifest hyperbolic dependence on oxygen concentration?

ROS generation and oxygen, ROS and [O2], Mitochondria, H2O2 production, reverse electron transfer, hypoxia, mitochondria, antimycin, State 3, State 4, respiration,

Figure: Mitochondrial respiratory chain in brain mitochondria. Complexes I-IV and glycerol 3-phosphate dehydrogenase (GPDH). the sites of ROS production are shown. In the absence of inhibitors, the dependence of H2O2 release on oxygen concentration is linear. Only in the presence of antimycin A the dependence is hyperbolic.

Figure: Possible sequence of events in conditions of ischemia or lack of oxygen. If oxygen is absent the A-form (A) spontaneously converts to the D-form (DSH), which can be re-activated back in the case of reoxygenation (given substrate ubiquinone availability). Depending on the particular conditions in situ the ND3 thiol group residue can be reversibly S-nitrosated by nitrosothiols (DSNO) or irreversibly oxidised by peroxynitrite or ROS (DS*). In the latter case the enzyme is irreversibly inhibited making this the initial step of mitochondrial damage in I/R. S-nitrosated enzyme (DSNO) can be reduced by mitochondrial glutathione and thioredoxin therefore further delaying the re-activation of the complex I at the early stages of reperfusion.

Figure: Effect of global ischemia on concentration of some TCA cycle metabolites in the heart (A) and brain (B) tissue. White and grey bars correspond to control and ischemic samples, respectively.

Complex I in Brain Ischemia

To date, ischemic stroke remains the third leading cause of death and devastating long-term neurological disability in industrialized countries, but the mechanisms of acute brain injury are still not completely understood. The loss of cerebral blood flow leads to decreased oxygen levels in the ischemic territory, initiating a sequence of pathophysiological events that cause ischemia/reperfusion (IR) damage. Oxidative stress and bioenergetic failure have long been recognized as an early event in the ischemic cascade and the primary cause of IR injury. However, the molecular details of mitochondria-mediated IR damage remain to be elucidated.

We are studying the ischemia/reperfusion-induced global changes of mitochondrial function and its spatio-temporal restoration in a mouse model of transient focal ischemia by transient occlusion of the middle cerebral artery (MCAo). Our studies provide a novel insight into understanding of the mechanisms underlying neuronal dysfunction in stroke. Ameliorating the cellular bioenergetics failure may serve as an attractive strategy in preventing subsequent detrimental events in the ischemic cascade, leading to reduced neuronal death after an ischemic stroke.

We found that ischemia induced a reversible loss of flavin mononucleotide from mitochondrial complex I leading to a transient decrease in its enzymatic activity, which is rapidly reversed on reoxygenation. Reestablishing blood flow led to a reversible oxidative modifcation of mitochondrial complex I thiol residues and inhibition of the enzyme. Administration of membrane permeable glutathione-ethyl ester at the onset of reperfusion prevented the decline of complex I activity and was associated with smaller infarct size and improved neurological outcome, suggesting that decreased oxidation of complex I thiols during I/R-induced oxidative stress may contribute to the neuroprotective effect of glutathione ester [Kahl et al., 2017; Stepanova et al., 2017]

Middle cerebral artery occlusion, ischemic stroke, mouse brain, Animal stroke model, mitochondrial Complex I, ischemic brain injury, ischemia/reperfusion

Figure: Mice are subjected to ischemia/reperfusion transient Middle Cerebral Artery occlusion (MCAo). At different time pointsafter reperfusion mice are sacrificed and tissue samples from the affected area are prepared. Mitochondrial respiration, citrate synthase activity and activities of respiratory chain enzymes are measured in whole tissue homogenates or isolated mitochondria.

Anaerobic Bioenergetics of Mitochondrial Complex I

The past decade has revealed a new role for mitochondria in cancer - regulation of oxidative phosphorylation/glycolytic pathways. Prominent features of cancer cells include changes in energy metabolism, mitochondrial alterations and enhanced resistance to apoptosis. The fact that some types of tumor use glycolysis and anaerobic respiration to meet their metabolic demands has been recognized by many researchers. Mitochondria of cancer cells associated with hypovascular tumors maintain energy-production in the absence of oxygen. It has been found that some types of cancer cells rely on the mitochondrial NADH:fumarate reductase system to generate energy. This peculiar reaction is catalyzed by complex I and complex II together when the latter is working in "reverse" mode. Thus, complex I reduces quinone to quinol and complex II oxidizes quinol by producing succinate from fumarate. Together these two enzymes catalyse  fumarate reduction by NADH so that complex I tranlocates protons and builds up transmembrane potential, which can be used by ATP synthase to form ATP (figure below, left). The key component of that system is mitochondrial Complex I for which the catalytic properties are not completely understood. At the same time, NADH:fumarate reaction catalyzed by bovine heart mitochondrial membranes is extremely slow (0.05% of standard physiological activity) and is inhibited by low concentration of succinate [Drose et al., 2016].

Cancer-induced bioenergetic features may be used  for the development of novel classes of anti-tumor agents. Our approach aims at the dissection of energy metabolism of cancer cells by targeting mitochondrial proteins and membranes in response to the hypoxia.

NADH:fumarate reductase, anaerobic respiration, cancer bioenergetics, succinate, reverse electron transfer

Figure: Possible metabolic pathway in which Complex I and Complex II) utilize fumarate resulting in anaerobic respiration. Complex I Q and QH2oxidized and reduced ubiquinone respectively. Proton-motive force generated in this reaction by Complex I potentially can be utilized by ATP-synthase (Complex V)