Alexander Galkin

Research

INTRODUCTION

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.

RESEARCH PROJECTS

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.

 

Hypoxic A/D Transition of Mitochondrial Complex I

Hypoxia is a lack of oxygen which is sufficient to cause cellular response of the tissue. Hypoxia can be caused by many factors including pathology or trauma, and it also occurs during surgery. Hypoxia is followed by restoration of oxygen supply, so-called 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 therefore to ATP-synthesis.  Complex I is known to undergo reversible conformational changes in situations in which its turnover is limited. As initially proposed in Andrei Vinogradov 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 catalyse 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 labelled 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 it’s 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 A/D transition in neonatal brain [Stepanova et al.,  2017]. Mitochondrially targeted nitrosothiol MitoSNO specifically modifies ND3 Cys-39 and results in attenuation of ischemia/reperfusion injury in neonatal brain [Kim et al., 2018]. 




A/D transition, Complex I, nitrosation, conformational change, ischemia, hypoxia, mitochondria

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.

Conformational change of mitochondrial Complex I

 

Video: Subunits involved into ischemic active/deactive (A/D) transition of mitochondrial complex I (based on PDB ID: 4WZ7). The protein is shown in grey and subunits involved into transition shown in colour using a cartoon representation (ND3 red, ND1 pink, PSST blue, 49 kDa beige, 39 kDa green). Iron-sulfur clusters are represented as yellow spheres. Schematic conformational change of partially resolved hydrophilic loop of ND3 THM1-2ND3 in the A and in the D-form is shown at the end of the clip. Subunits involved into ischemic active/deactive (A/D) transition of mitochondrial complex I (based on PDB ID: 4WZ7). The protein is shown in grey and subunits involved into transition shown in using a cartoon representation (ND3 red, ND1 pink, PSST blue, 49 kDa beige, 39 kDa green). Iron-sulfur clusters are represented as yellow spheres. Schematic conformational change of partially resolved hydrophilic loop of ND3 THM1-2ND3 in the A and in the D-form is shown at the end of the clip.

 

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 further confirmed after the structure of both forms was published by Hirst group [Agip et al., 2018] (PDB structure of the deactive and active enzyme from mouse are available now). The structural and functional studies of the A/D transition were initiated in the 90's [Kotlyar&Vinogradov, 1990; Maklashina et al., 1994], continued in our lab and currently under investigation in several laboratories worldwide.




 

Oxaloacetate Inhibition of Succinate Dehydrogenase (Complex II)

Mitochondrial Complex II or succinate dehydrogenase (SDH) is a key mitochondrial enzyme connecting the tricarboxylic acid (TCA) cycle and the electron transport chain. Studies of complex II are clinically important since new roles for this enzyme have recently emerged in cell signalling, cancer biology, immune response, and neurodegeneration. Oxaloacetate (OAA) is an intermediate of the TCA cycle and at the same time is an inhibitor of complex II with high affinity (Kd~10-8 M). Whether or not OAA inhibition of complex II is a physiologically relevant process is a significant, but still controversial topic. We found that complex II from mouse heart and brain tissue has similar affinity to OAA and that only a fraction of the enzyme in isolated mitochondrial membranes (30 and 55% in the heart and brain, respectively) is in the active form. Since OAA could bind to complex II during isolation, we established a novel approach to deplete OAA in the homogenates at the early stages of isolation. In the heart, this treatment significantly increased the fraction of free enzyme, indicating that OAA binds to complex II during isolation. In the brain the OAA-depleting system did not significantly change the amount of free enzyme, indicating that a large fraction of complex II is already in the OAA-bound inactive form.   Our data show that in brain OAA is an endogenous effector of complex II, potentially capable of modulating the activity of the enzyme.

Furthermore, short-term ischemia resulted in a dramatic decline of OAA in tissues (figure below, control and ischemia white and grey bars, respectively), but it did not change the amount of active complex II.  On the one hand, it could indicate that despite a sharp decrease in OAA tissue concentration, the inhibitor is still bound to the enzyme because of its very slow rate of dissociation (0.02 min−1). Another possibility is that the inactivating OAA-induced conformational change in SDH may occur after the binding of the inhibitor, and the subsequent removal of OAA does not activate the enzyme. Therefore, the state of SDH in situ is determined by the conformational change of the enzyme rather than OAA concentration in the matrix per se. Conformational change of mammalian succinate dehydrogenase  is currently being investigated in our laboratory [Stepanova et al., 2016].

Oxaloacetate in ischemia, TCA cycle in ischemia, Krebs cycle Complex I in stroke

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 QH2 – oxidized and reduced ubiquinone respectively. Proton-motive force generated in this reaction by Complex I potentially can be utilized by ATP-synthase (Complex V)