Mitochondrial ATP is produced by the oxidative phosphorylation machinery. OXPHOS couples the phosphorylation of ADP and electron transfer through a chain of oxidoreductase reactions. These oxidoreductase reactions are carried out by five enzymes/enzyme complexes in the IMM: NADH:Ubiquinone oxidoreductase (Complex I, CI), Succinate:Ubiquinone oxidoreductase (Complex II, CII), Ubiquinol:Cytochrome c oxidoreductase (Complex III, CIII), Cytochrome C oxidase (Complex IV, CIV) and the ATP synthase (Complex V, CV). Complex I is the biggest of five respiratory complexes and CII is the smallest. Each of these five complexes is essential and four of the five complexes CI thru CIV pump a total of 5 protons from the matrix to the intermembrane space per pump cycle, which is essential for the maintenance of the mitochondrial membrane potential. The protons flow back via CV, which acts in a reverse mode producing ATP as opposed to the normal mode where ATP hydrolysis feeds transport. In addition to their role in OXPHOS, the complexes also play important roles in generation of ROS which can either be deleterious or beneficial, based on the context and extent of their generation.
Eukaryotic CI is located in the IMM protruding into the matrix to form an “L” shaped structure. Forty five subunits are identified as comprising the bovine CI, which is closely related to human CI195. The subunits are named according to their apparent molecular weights (75, 51, 49, 30 and 24 kDa subunits), or for the first four amino acids of the mature protein sequences (PSST and TYKY) or for the NADH dehydrogenase products of the mitochondrial DNA (ND1 to ND6 and ND4L) 14 of these subunits are conserved and are sufficient for energy transduction195. The other
subunits differ from species to species. The core conserved subunits form two domains supported by the supernumerary subunits. Seven of the 14 subunits are hydrophilic and constitute the redox domain and are present in the matrix and the 7 hydrophobic units are present in the IMM196,197. In eukaryotes, the hydrophobic subunits are mitochondrial encoded whereas the supernumerary units are nuclear encoded198,199. The seven hydrophilic subunits form a Y shaped domain encasing the cofactor cohort of complex I: a flavin nucleotide for NADH oxidation and a chain of 7 Fe-S clusters (one [2Fe-2S] and 6 [4Fe-4S] - labeled N1 thru N7, though not necessarily present in that order)196. The 7 clusters are split on either side of the flavin moiety with the 2Fe-2S cluster lying
34 electrons to the quinone200. Subunits ND2, ND4 and ND5 are structurally similar and are related to the subunits of Mrp family of Na+/H+ antiporters indicating that they are likely sites of proton
transfer across the membrane. These subunits are at a significant distance away from the FeS cluster chain and the quinine binding site. Hence coupling of the redox and transport processes require long range energy transfer through the protein201. This long range energy transfer forms a key factor in the generation of ROS especially in IR given a lack of receptor oxygen and availability of free electrons, which in turn react with any available oxygen forming ROS and nitrogen moieties forming reactive nitrogen species (RNS). Excess ROS and RNS generation is a key deleterious event in IR injury.
Complex I in ischemia-reperfusion
Acidification of cytosol following ischemia has been shown to be the key factor behind the blockage of complex I202. This blockage of complex I is a precursor of mitochondrial dysfunction following IR. The consequence of blockade of complex I, during reperfusion is an increase in the production of ROS, which ultimately leads to the opening of MPTP, following various pathways such as post-translational modifications induced by ROS and ROS induced dysfunction of ion channels leading to Ca2+ overload, and the induction of cell death. Superoxide anion production by antimycin inhibited bovine heart mitochondrial particles with NADH was first reported by Turrens et al35. The site of this production was deduced as the respiratory complex I based on the observation that superoxide generation was reduced in the presence of rotenone- a complex I blocker. Takeshige et al. showed for the first time that complex I was indeed the site of formation of NADH and NADPH-dependent superoxide formation203. Kang et al. showed differential
kinetics of superoxide formation by complex I with NADH dependent reactions being much faster than those induced by NADPH204. Hence numerous studies have focused on the beneficial effect of partial or complete blockage of complex I for decrease in ROS during the critical initial phase of reperfusion where there is an increase in oxygen availability.
Although complex I itself is a major source of ROS, complex III is the principal site of superoxide generation during oxidation of complex I substrates and hence the hypothesis that blockage of complex I essentially depletes complex III of the electrons needed for the generation of superoxide,
35 protecting the mitochondria and consequently the heart. This blockage can be achieved during ischemia or reperfusion. Blockade of electron transport at complex I by rotenone immediately before ischemia preserves respiration through cytochrome oxidase in the distal electron transport chain and significantly reduces cytochrome c loss from the mitochondria during ischemia205. As previously stated, blockage of complex I using rotenone can be a protective strategy in IR. However, since rotenone is a non-reversible blocker of complex I, this is not a permissible strategy as it would compromise resumption of normal OXPHOS needed for the recovery of cellular function. Hence Chen et al. used amobarbital, a reversible complex I blocker to block complex I activity immediately before IR and observed a significant reduction in infarct size, and decreased H2O2 and cytochrome c release206. In further studies, Aldakkak et al. found that even though
administration of amobarbital at ischemia increased superoxide and NADH levels and decreased mitochondrial Ca2+, during ischemia, superoxide levels and mitochondrial Ca2+ were lower and ultimately a decrease in infarct size- consistent with a decrease in complex I activity and consequent better preservation of cardiac tissue207. Xu et al. also showed that a transient blockage of complex I activity achieved by extracellular acidification at the onset of reperfusion also led to a better preservation of tissue following ischemic injury202. The protective effect of the blockage of complex I is also observed in aging induced myocardial injury and mitochondrial dysfunction208.
36