Сitrate synthase catalyzes the first step in the TCA cycle: transfer of the acetate group of acetyl-CoA to oxaloacetate, producing citrate. The enzyme is a homodimer composed of two 50-kDa subunits. Each subunit contains two domains separated by a deep cleft forming the active site of the enzyme. A number of substances, including succinyl-coenzyme A and adenine nucleotides, are inhibitors of citrate synthase.

Aconitase catalyzes the reversible isomerization of citrate and isocitrate via cis-aconitate. This process involves four stereospecific reactions: the dehydration of citrate or isocitrate to cis-aconitate and the rehydration of cis-aconitate to isocitrate or citrate. Mitochondrial aconitase is a monomeric 83-kDa protein containing labile [4Fe–4S]²⁺ cluster.

The aconitase plays a role in oxidative stress: it shows a high sensitivity to H₂O₂, being completely inactivated at very low H₂O₂ concentrations (less than 50 μM) and other ROS [36, 37], either due to interaction with the iron–sulfur cluster [38, 39] or indirectly via interaction between the enzyme and a membrane component responsive to peroxide [39]. Inhibition of aconitase could lower the flux through the cycle due to accumulation of citrate and a consequent inhibition of citrate synthase. Yarian et al. [40] have demonstrated that the activity of aconitase is altered in rats during the aging process. They suggested that an age-related decrease in aconitase activity may be associated with an age-related increase in the prooxidant state in mitochondria and is likely to contribute to a decline in the overall efficiency of mitochondrial bioenergetics.

Mammalian NAD isocitrate dehydrogenase exists primarily as an octamer of composition (α₂βγ)₂. Three different subunits of the enzyme are structurally related and have molecular masses around 40 kDa [41, 42]. Substrate binding and active sites are shared between the subunits [43, 44]. The enzyme plays a controlling role in the formation of NADH for the respiratory chain and shows complex regulatory properties. In particular, the enzyme is inhibited by increasing ATP/ADP and NADH/NAD⁺ ratios. Calcium ions activate mammalian NAD isocitrate dehydrogenase, and the enzyme is more sensitive to Ca²⁺ at lower ATP/ADP ratios [45, 46].

Alternatively, isocitrate can be converted to 2-oxoglutarate in mitochondria by NADP isocitrate dehydrogenase [45], and reducing equivalents from produced NADPH can be transferred to NAD⁺ by nicotinamide transhydrogenase localized in the MIM, to give NADH [47]. NADP isocitrate dehydrogenase shows no allosteric regulation [45]. Normally, it is the regulated NAD⁺-linked enzyme that participates in the TCA cycle [48].

Similarly to PDHC, 2-oxoglutarate dehydrogenase is a multienzyme complex (OGDHC) performing oxidative decarboxylation of 2-oxoglutarate (OG). Chemical reaction catalyzed by OGDHC proceeds via the sequential steps catalyzed by protein components of OGDHC, 2-oxoglutarate dehydrogenase (E1), dihydrolipoyl succinyl transferase (E2), and dihydrolipoyl dehydrogenase (E3). E1 performs the first two reactions: a decarboxylation of 2-oxoglutarate and a reductive succinylation of lipoic acid. E1 contains coenzyme molecule (thiamine diphosphate, TDP) and Mg²⁺ as a cofactor. First, TDP in the active center of E1 decarboxylates 2-oxoglutarate, and hydroxybutyrate fragment covalently attaches to TDP. The carbon dioxide produced by this reaction is nonpolar and small and can diffuse out of the mitochondria and out of the cell. Then E1 catalyzes reductive succinylation of lipoic acid: transfer of succinyl from TDP to lipoic acid (cofactor covalently bound to E2). E1 has the lowest catalytic activity among the components and as such limits the rate of the overall OGDHC-catalyzed process [49]. As with pyruvate dehydrogenase, it is likely that the step catalyzed by E1 normally regulates the overall OGDHC activity [50].

E2 is the second of the three enzyme components participating in the conversion of 2-oxoglutarate into succinyl-CoA. E2 catalyzes transfer of succinyl group from lipoic acid to CoA. Thus, E2 produces the end product of 2-oxoglutarate decarboxylation, succinyl-CoA, and reduced form of lipoic acid, dihydrolipoamide. Succinyl-CoA released from the E2, continues further degradation within the TCA cycle.

E3 is the same flavoprotein as the one contained in the PDHC (see above) and serves same functions—regeneration of the cofactor on E2 (by conversion of dihydrolipoamide into lipoamide) and production of NADH.

All enzymes, participating in the conversion of 2-oxoglutarate into succinyl-CoA, are organized into multimeric complex, OGDHC. The central OGDHC hollow is made of predominantly 24 E2 molecules, to which are attached dimeric E1 and E3 subunits (the latter attachment may be also via binding to E1 subunits) [51, 52]. Each OGDHC probably contains six E1 dimers.

As with pyruvate dehydrogenase, the 2-oxoglutarate dehydrogenase is end product inhibited by increases in the succinyl-CoA/CoA, NADH/NAD⁺, and ATP/ADP ratios. However, unlike pyruvate dehydrogenase, the enzyme is not regulated by reversible phosphorylation. 2-oxoglutarate dehydrogenase is directly activated by Ca²⁺, and decreases in ATP/ADP ratio markedly increase the sensitivity of the enzyme to calcium ions [46, 50]. Unlike isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase is an order of magnitude more sensitive to activation by Ca²⁺. OGDH has been found to be inhibited in cardiac mitochondria under myocardial ischemia and reperfusion [53]. This inactivation involves ROS-dependent oxidative damage of lipoic acid essential for the catalytic activity of E2.

It seems worth to note that OGDHC also catalyzes several side reactions of potential biological significance. Under the conditions of excessive accumulation of the dihydrolipoamide intermediate, OGDHC catalyzes oxidation of the latter by molecular oxygen with the formation of the ROS (superoxide anion radical) and thiyl radical of the E2-bound lipoamide [54]. OGDHC-dependent ROS may contribute to cellular signaling and/or damage. Another biologically relevant side reaction of OGDHC is the carboligase activity of its first TDP-dependent E1 component, which appears to be involved in glyoxylate scavenging [55].

Succinate dehydrogenase (SDH) is a unique enzyme participating in both the TCA cycle and respiratory electron transfer chain, OXPHOS (Fig. 5.5). In this section, we focus on the functioning and the role of SDH in the citric acid cycle, whereas involvement of it in the ETC will be described later.

Within the citric acid cycle, SDH oxidizes succinate to fumarate. Eukaryotic enzyme consists of four subunits. For instance, the porcine cardiac SDH consists of a hydrophilic head that protrudes into the mitochondrial matrix compartment and a hydrophobic tail that is embedded in the MIM [56, 57]. The head consists of two subunits (SdhA, SdhB in mammals) forming the catalytic core. SdhA and SdhB subunits contain the redox cofactors that participate in ETC. 70-kDa SdhA contains the covalently bound FAD cofactor and the binding site for succinate. 27-kDa SdhB contains the three Fe–S centers that mediate electron transfer to ubiquinone [57, 58]. SdhB also forms the interface between the catalytic domain and the membrane domain of the complex (15-kDa SdhC and 12–13-kDa SdhD subunits with heme b binding in mammals).

Oxidation of succinate in the active center of SdhA subunit involves the two-electron-dependent reduction of FAD. Then electrons from FADH₂ travel via Fe–S centers of SdhB subunit to the ubiquinone (coenzyme Q, CoQ) bound to Q_P and Q_D sites formed by SdhC and SdhD subunits and reduce it to ubiquinol. Thus, SDH links TCA cycle (succinate oxidation) to ETC (ubiquinol formation).

The catalytic activity of SDH is modulated by posttranslational modifications (phosphorylation, acetylation) and by active site inhibition. In mouse SdhA, reversible acetylation at Lys residues was shown to attenuate catalytic activity of SdhA [59]. The level of SdhA acetylation is controlled by deacetylase SIRT3. In mammalian cells the SdhA subunit is phosphorylated and this modification, like acetylation, decreases enzymatic activity [60]. SdhA is also a substrate of the tyrosine kinase Fgr in vitro, although the physiological significance of this modification is not known [61]. SdhA activity is markedly decreased in the post-ischemic myocardium, and this loss of activity is associated with another covalent modification—deglutathionylation of Cys-90. It has been suggested that deglutathionylation facilitates sensitivity of the 70-kDa SdhA subunit to oxidative stress induced by ROS during post-ischemic reperfusion of myocardium [62]. SDH catalytic activity is modulated by TCA-cycle intermediates including oxaloacetate, which is a potent inhibitor. Succinate and ATP facilitate the dissociation of oxaloacetate from SDH thereby activating the enzyme. The oxaloacetate-dependent inhibition contributes to the modulation of SDH activity by the metabolic status of mitochondria [63].

Mutations in SdhA and SdhB impair SDH activity and cause diseases. For example, mutations in SdhA are associated with several types of neurodegenerative disorder in humans (subacute necrotizing encephalomyelopathy, SNEM, also known as Leigh syndrome) [64–67] and with symptoms of late-onset myopathy [68]. It has been speculated that the mutation could alter SdhA’s conformation or redox state which might increase sensitivity of SdhA to the inhibitory effect of oxaloacetate [64]. Mutations in SdhB are associated with paragangliomas 4 (PGL4s)—neuroendocrine tumors that can occur in cells of the neural crest commonly located adjacent to the carotid body, as well as in the adrenal glands (there they are called pheochromocytomas, PCCs) [69–71]. Mechanism of tumor formation may include accumulation of succinate due to dysfunction of SDH [72], succinate-dependent inhibition of cytosolic prolyl hydroxylase (PHD), and the accumulation of hypoxia-inducible factor (HIF) by preventing its degradation and resulting in enhancement of tumorigenesis through the resistance to apoptotic signals [73]. Also the increase in HIF can lead to the stimulation of angiogenesis that will feed and supply the tumor [74].

The active form of fumarate hydratase (FH) is a homotetramer in which three of the four subunits form the enzymatic active site for the reversible conversion of fumarate to malate. Dysfunction of FH leads to the accumulation of fumarate in the mitochondria [72]. Similar to succinate, increased fumarate levels cause in the cytosol series of events that enhance tumorigenesis [72–74]. In the case of FH deficiency, accumulation of intramitochondrial fumarate has been shown to slow down the SDH reaction in skeletal muscle [75].

This enzyme catalyzes the conversion of malate into oxaloacetate. Important malate dehydrogenase (MDH)-catalyzed co-reaction is the production of high-energy equivalent, NADH, which serves as a fuel for mitochondrial OXPHOS. There are two main isoforms of MDH in eukaryotic cells, soluble (MDH1) and mitochondrial (MDH2), both being homodimers of 30–35-kDa subunits. MDH contains two distinct domains, the amino-terminal NAD-binding domain and the carboxy-terminal domain with substrate binding site. The active site of the enzyme is in a cleft between two domains [76]. Mitochondrial MDH2 is highly regulated by TCA-cycle products: high malate concentrations stimulate activity of MDH2, whereas high oxaloacetate concentrations inhibit the enzyme. In addition, citrate can allosterically inhibit MDH2-dependent oxidation of malate when levels of malate and NAD⁺ are low and stimulate MDH2 in the presence of high levels of malate and NAD⁺ [77, 78]. According to Mullinax et al. [78], all three effectors bind to the same putative allosteric site on MDH2. Mitochondrial enzyme takes part in the TCA cycle under conditions where the conversion of malate into oxaloacetate is unfavorable (under standard conditions, the MDH-catalyzed reaction tends to form malate). This problem is solved via formation of complex of MDH2 with fumarase and citrate synthase where the removal of oxaloacetate from MDH is facilitated toward citrate synthase, so the TCA cycle is driven toward the oxaloacetate formation [79].

Recently, Wang et al. [80] have reported specific interaction of MDH2 with several subunits of OXPHOS Complex III (bc1 complex, or coenzyme Q: cytochrome c—oxidoreductase) in the heart. Association of MDH is strong (K _D is 0.047 μM) and includes interaction with two core subunits (1, 2) and N terminus of subunit 5 (Rieske iron–sulfur protein). Functional consequences of MDH2 binding to Complex III include enhancement of the bc1-activity and activation of MDH2. Interestingly, only reverse reaction (the reduction of oxaloacetate to malate) is stimulated by Complex III. It is suggested that cross talk between Complex III and MDH2 can lead to the increased transport of malate from mitochondria to the cytosol, where it can be converted to oxaloacetate by MDH1 and enter gluconeogenesis.

Soluble isoform of malate dehydrogenase, MDH1, participates in the malate–aspartate shuttle with exchanging reducing equivalents so that malate can pass through the mitochondrial membrane and be transformed into oxaloacetate for further cellular processes. Reducing equivalents are produced in the cytosol during glycolysis in the form of NADH. In order to be utilized in OXPHOS, they have to be transported to mitochondria. However, the MIM is impermeable to NADH, and malate shuttle mechanism exists to carry reducing equivalents across the membrane in the form of malate. MDH1 localized in the mitochondrial intermembrane space oxidizes cytosolic NADH to convert oxoloacetate to malate. Malate is imported into the mitochondrial matrix by malate–oxoglutarate antiporter. In the mitochondrial matrix, malate is converted by mitochondrial MDH into oxaloacetate (see above), during which NAD⁺ is reduced with two electrons to form NADH and release an H⁺. As a result, NADH in the mitochondria can be used for OXPHOS to synthesize ATP. MDH1 is highly expressed in cardiac and skeletal muscles where it functions as a part of malate shuttle system for aerobic energy production for muscle contraction [81]. Interestingly, levels of MDH1 increase in the left ventricle of dilated cardiomyopathy patients indicating that MDH1 is involved in the activation of glucose oxidation in hypoxic environment of hypertrophied myocardium [81].

At the end of each TCA cycle, oxaloacetate is regenerated, and the cycle can continue. The NADH generated in the TCA cycle donates electrons in OXPHOS to synthesize ATP. Similarly, FADH₂ donates electrons to coenzyme Q, which is an intermediate in the ETC. For each acetyl group entering the TCA cycle in the form of acetyl-CoA, three molecules of NADH, one molecule of ubiquinol, and one molecule of GTP are produced.

Complex I is the primary entry point of electrons into the ETC: at this step, electrons from NADH are transferred to ubiquinone. NADH oxidation regenerates NAD⁺ for the TCA cycle and FAO. Potential energy of two electrons transferred from NADH to ubiquinone is used by CI to transport protons across the MIM (four protons are translocated for each two-electron oxidation of NADH). Thus, CI contributes to the development of electrochemical proton gradient.

Net reaction catalyzed by CI:

CI is the large multiprotein complex (∼1 MDa) composed of 45 subunits [82, 83]. It is “L” shaped, having two “arms” perpendicular to each other. A hydrophobic “arm” is embedded in the MIM, and a hydrophilic peripheral “arm” protrudes into the mitochondrial matrix [84]. Functionally, there are three modules in CI: the dehydrogenase module composed of the subunits NDUFV2, NDUFV1, and NDUFS1, responsible for the oxidation of NADH to NAD⁺; the module composed of the subunits NDUFS2, NDUFS3, NDUFS7, and NDUFS8, responsible for transfer of the electron to ubiquinone; and the proton translocation module composed of the seven subunits and located within the membrane “arm” [84–86].

Within the hydrophilic “arm,” one domain is formed by NDUFV1 and NDUFV2. This domain is the place where NADH-binding site and flavin mononucleotide (FMN) are located (both on NDUFV1). Chain of iron–sulfur clusters also starts in this domain. In particular, the iron–sulfur clusters closest to FMN are [2Fe–2S] and [4Fe–4S] non-covalently bound to NDUFV2 and NDUFV1, respectively. This chain continues through another domain formed by [2Fe–2S]- and [4Fe–4S]-containing NDUFS1 and ends in the lower part of hydrophilic “arm” built of NDUFS2, NDUFS3, NDUFS7, and NDUFS8. There are another three [4Fe–4S] iron–sulfur clusters in this part of CI: two of them are non-covalently attached to NDUFS8, and the most distal cluster is attached to NDUFS7. Although the location of ubiquinone-binding site remains unknown, on the basis of site-directed mutagenesis of the NDUFS2 and NDUFS7 subunits, the putative site is usually placed at the interface of NDUFS2 and NDUFS7 subunits in the close proximity to the [4Fe–4S] iron–sulfur cluster on NDUFS7 [87, 88].

Lower part of the hydrophilic “arm” includes the interface with the membrane domain of CI, the hydrophobic “arm.” The latter one is composed of seven highly hydrophobic core subunits—ND1–ND6 and ND4L. Subunits ND4, ND5, and ND2 are related to the Mrp family of Na⁺/H⁺ antiporters, and they are often suggested to participate in proton translocation [89].

Prime function of CI upon the binding of NADH is oxidation of NADH by FMN. During oxidation a pair of electrons is transferred from NADH to the flavin. Then they are transferred (one at a time) across the chain of seven iron–sulfur clusters and reunion on a ubiquinone, thus reducing it to ubiquinol. Ubiquinol then dissociates. Ubiquinone (coenzyme Q₁₀, CoQ₁₀) is fat-soluble mobile in mitochondrial membrane molecule. It plays a unique role in the ETC as a carrier of electrons from CI and CII to CIII (see below). At some unknown point, transfer of the electrons from NADH to ubiquinone is coupled to translocation of protons across the membrane.

Under certain conditions an electron of reduced flavin may “escape” from intramolecular traveling through iron–sulfur clusters and hijack a molecule of O₂. This results in superoxide production by CI [90]. In addition, [4Fe–4S] iron–sulfur cluster on NDUFS7 subunit as well as semiquinone contributes to ROS production by CI [91].

A number of genetic abnormalities in various subunits and assembly factors of CI lead to the CI deficiency. CI deficiency is the most common cause of mitochondrial disease in both children and adults, and main clinical phenotypes include Leigh syndrome, fatal infantile lactic acidosis (FILA), neonatal cardiomyopathy, leukoencephalopathy, pure myopathy, and combined hepatopathy and tubulopathy [92–95].

Succinate–ubiquinone oxidoreductase (SDH or CII) is a second entry place for electrons from high-energy equivalents formed in mitochondria during the oxidation of pyruvate, FAs, and amino acids. Specifically, the electron transfer pathway from CII-catalyzed oxidated succinate involves initial reduction of a FAD cofactor followed by electron transfer through three Fe–S centers to ubiquinone.

Reactions catalyzed by SDH/CII:

CII is a part of both the TCA cycle and ETC (Fig. 5.5). Eukaryotic CII consists of four subunits and is the only ETC component not pumping protons across the MIM during catalytic cycle. There are two major structural components in the CII: the hydrophilic head formed by SdhA and SdhB, which catalyzes oxidation of succinate during TCA cycle, and a hydrophobic tail mostly embedded in the MIM. We already described the functioning of hydrophilic head (see Production of Reducing Equivalents: Tricarboxylic Acid Cycle), so we concentrate here on the hydrophobic compartment participating in the ETC (to be precise, delivery of electrons from FADH₂ to ETC is initiated in the hydrophilic subunit SdhB containing the three Fe–S centers that mediate electron transfer to ubiquinone).

The hydrophobic membrane domain of CII consists of two subunits (SdhC, SdhD). These subunits contain a bound heme b moiety and two ubiquinone-binding sites [57]. The high-affinity ubiquinone site (proximal, Q_P) is the site where ubiquinone reduction occurs in two stepwise single-electron reactions. Q_P site stabilizes the partially reduced semiquinone thus preventing dissociation of this potential ROS generator and favoring release of fully reduced ubiquinol [56]. The second site in the hydrophobic domain of CII (distal, Q_D) has low affinity for ubiquinone. Ubiquinone may undergo reduction at the Q_D site via electron transfer from the heme, although proof of this is lacking. The significance of the heme moiety and Q_D site in CII remains unclear.

Mutations in the structural subunits and in factors regulating assembly and activity of CII cause a variety of human diseases [96–98].

CIII receives electrons from upstream electron transfer complexes (CI and CII) in the form of reduced coenzyme Q₁₀, ubiquinol (QH₂). CIII is a dimer embedded in the MIM. In mammals, each monomer is composed of 11 different structural subunits [99]. Three subunits of Complex III contain the metal centers responsible for electron transfer: cytochrome b (subunit MTCYB) contains two b-type heme groups, b _L and b _H; cytochrome c1 (subunit CYC1) contains heme group c; and Rieske iron–sulfur protein (subunit UQCRFS1, or RISP) contains [2Fe–2S] cluster-binding domain. The function of the other eight subunits remains to be elucidated. CIII catalyzes transfer of electrons from QH₂ to cytochrome c (cyt-c). The released energy is used to expel protons from the mitochondrial matrix to the intermembrane space, resulting in the generation of a proton gradient across the MIM. Finally, cytochrome c carries the electrons from CIII to cytochrome c oxidase (CIV).

Three subunits form the intermembrane side of CIII monomer. In particular, it contains the functional domains of cytochrome c1 and the RISP, as well as subunit 8. The transmembrane region of monomer contains membrane-spanning components: cytochrome b; subunits 7, 10, and 11; and the transmembrane helices of the RISP and cytochrome c1. Finally, the side of the CIII which faces mitochondrial matrix is composed of subunits 1, 2, 6, and 9.

According to Q-cycle mechanism, CIII-catalyzed hydroquinone oxidation starts with the binding of QH₂ in the intermembrane side of CIII (positive side, Q_P). Importantly, before QH₂ can be oxidized, it must first be deprotonated by releasing two protons into mitochondrial intermembrane space. Each QH₂ molecule is a donor of two electrons. One of them is transferred to the 2Fe–2S cluster of RISP. The reduction of the Rieske center increases the affinity of the CIII for semiquinone by several orders of magnitude, stabilizing the semiquinone radical at the Q_P site. Electron from reduced 2Fe–2S cluster of RISP is transferred to the iron of heme C-containing cytochrome c1. The latter is a 30-kDa subunit of CIII serving as an electron donor to the iron of heme C-containing highly soluble cytochrome c for further transportation to Complex IV. Electron pathway bifurcates during full oxidation of QH₂ at the Q_P site: second electron is accepted by b _L heme of cytochrome b (not by RISP). From b _L heme this electron travels to b _H heme, and finally it becomes accepted by quinone located in the matrix side of CIII (negative side, Q_N). Complete reduction of quinone at the Q_N site requires two electrons, so second chain of reactions starting from second QH₂ at the Q_P site completes the whole process. It is important to mention that two protons are recruited by CIII to protonate reduced quinone in the Q_N site.

Thus, the following processes take place during CIII-dependent electron transfer from ubiquinol to cytochrome c:

With the resulting net equation:

A small fraction of electrons can escape the electron transport chain before reaching CIV and reduce oxygen. Premature electron leakage to oxygen results in the formation of superoxide. Electron leakage occurs mainly at the Q_P site, when semiquinone reacts with oxygen to form superoxide and other ROS.

Recent evidence demonstrates novel cross talk between CIII and TCA-cycle enzyme, mitochondrial MDH. MDH interacts with CIII subunits 1 and 2 and RISP and can be coprecipitated with bc1 complex. Interaction of bc1 complex with MDH results in an increase of the activities of both the bc1 complex and MDH. Interestingly, CIII-dependent activation of MDH is unidirectional (oxaloacetate  →  malate). Cross talk between ETC component (CIII) and TCA-cycle enzyme (MDH) might play a regulatory role in mitochondrial bioenergetics [80].

Mutations in the cytochrome b cause CIII deficiency and disorders such as mitochondrial encephalomyopathy, cardiomyopathy, septo-optic dysplasia, and multisystem disorder [100–103]. Similarly, in a murine model of myocarditis (chagasic disease), mice infected with Trypanosoma cruzi demonstrate compromised ATP production in the myocardium because of a decline in cytochrome b expression. Myocardial bioenergetics is disturbed under pathological conditions because of inefficient electron transport for ATP synthesis in mitochondria. Loss in CIII activity can be improved by treatment of infected mice with antioxidant (phenyl-α-tert-butyl nitrone) which restores cytochrome b expression [104]. In addition, a deletion in ubiquinone-binding protein (subunit 7 or QP-C subunit) has been shown to underlie hypoglycemia and liver dysfunction [105].

CIV is the terminal enzyme of the mitochondrial ETC. It catalyzes the electron transfer from reduced cytochrome c to molecular oxygen to form water. The energy released in the reaction of oxygen reduction is conserved in the form of a transmembrane electrochemical gradient of protons across the MIM. Oxidation of every O₂ molecule requires four molecules of cytochrome c. The energy released during transfer of electrons from reduced cytochromes c to O₂ is used by CIV for net transfer of four protons from the matrix into the MIM, resulting in a proton gradient across the MIM. The overall reaction done by CIV can be described by the following equation:

Mammalian CIV is a heteromeric complex composed of 13 different subunits [106]. The structure of this complex has been resolved at present with resolution up to 1.8 Å. The catalytic core of the enzyme is formed by subunits MTCO1 and MTCO2, containing two heme group a moieties (cytochromes a and a ₃ ) and the two copper centers (Cu_A and Cu_B). Hemes a and copper centers are organized into two binuclear centers (a/Cu_A and a ₃ /Cu_B) responsible for the electron transfer. Heme a is an essential component of the catalytic centers of CIV found in all eukaryotic and some prokaryotic COX species [107]. More specifically, a ₃ /Cu_B binuclear center and heme a are located on MTCO1, whereas copper center Cu_A is located on MTCO2 almost on the border with subunit MTCO1. The chemistry of oxygen reduction to water occurs in the a ₃ /Cu_B catalytic site. Subunit MTCO3 is part of the structural core and may play a role in proton pumping.