Electron Transport System(ETS) and Oxidative Phosphorylation

The metabolic pathway through which the electron passes from one carrier to another, is called the electron transport system (ETS).In cellular respiration the energy of oxidation-reduction utilised for the conversion of ADP to ATP. It is for this reason that the process is called oxidative phosphorylation (Oxphos).

1.0Electron transport system(ETS)

In ETS, electrons flow through the chain in a stepwise manner from more electronegative compounds to more electropositive compounds.The last step in aerobic respiration is the oxidation of reduced coenzymes produced in glycolysis and Krebs' cycle by molecular oxygen through FAD, UQ (Ubiquinone), cytochrome b, cyto-chrome, c cytochrome a and cytochrome a, (cytochrome oxidase).

It is now well established that the electron transport chain or system in mitochondria consists of four multi-protein complexes (I to IV) which are localized in the inner mitochondrial membrane and also ubiquinone (UQ or coenzyme Q) and cytochrome-c which are not tightly bound to membrane protein but act as mobile carriers between the complexes.

2.0Complexes or Components of ETS

The composition of the mitochondrial electron transport system is basically similar in most living organisms although there may be some minor variations in the nature of some of the components among groups of organisms. 

The mitochondrial electron transport chain (ETC) includes complexes I‑IV, as well as the electron transporters ubiquinone and cytochrome c. 

There are two electron transport pathways in the ETC: Complex I/III/IV, with NADH as the substrate and complex II/III/IV, with succinic acid as the substrate. 

The electron flow is coupled with the generation of a proton gradient across the inner membrane and the energy accumulated in the proton gradient is used by complex V (ATP synthase) to produce ATP from ADP.

1. Complex I (CI)

Complex I (CI) is also called NADH-ubiquinone oxidoreductase complex, is the largest multisubunit enzyme complex in the ETC. The key role of CI is to transfer electrons from matrix NADH to ubiquinone, as the name implies. 

Mitochondria from the Bos taurus heart have been regarded as the best model for human Complex I . 

These studies demonstrate that the L-shaped eukaryotic Complex I contains two domains: 

The membrane arm embedded in the inner membranes and the matrix arm protruding into the matrix. The two domains are mainly composed of 14 core subunits that are conserved from bacterial Complex I and are the core of the enzymatic reaction. There are 45 clearly identified proteins that participate in the formation of the core subunits. The matrix arm contains seven core subunits that contain the following cofac-tors: 

A flavin mononucleotide (FMN) molecule; Seven to Nine FeS clusters [including the (2Fe-2S)_N1b, (4Fe-4S)_N3, (4Fe-4S)_N4, (4Fe-4S)_N5, (4Fe-4S)_N6a/b and (4Fe-4S)_N2 clusters]; and the final electron accepting iron-sulfur cluster (N2 cluster), which was recently found to deliver electrons to ubiquinone binding sites. 

The membrane arm contains seven hydrophobic subunits, all of which are encoded by the mitochondrial genome. An FMN bound at the cusp of the matrix arm could form FMNH₂ by accepting a pair of electrons derived from matrix NADH, which is primarily produced by the tricarboxylic acid (Krebs/TCA cycle that continuously occurs in the matrix. These interactions also mean that electrons go into the ETC and are then passed to ubiquinone via a chain of iron-sulfur clusters arranged from low to high potential. 

The ubiquinone binding site is located at the junction of the membrane arm and matrix arm, in which ubiquinone (CoQ) is reduced to ubiquinol (QH₂). 

NADH + UQ +H+ ⇆NAD + +UQH2

Then, the conformational changes induce the formation of a proton translocation channel by subunits near the CoQ binding site. The energy released by the transfer of a pair of electrons from NADH to CoQ in Complex I probably induce the pumping of four protons from the matrix into the intermembrane space. 

Several hypotheses exist in current research: Ohnishi proposed a hypothesis that two protons are indirectly pumped out in a conformation-coupled manner and that the other two protons are directly pumped out by the induction of ubiquinone redox. 

Sazanov and Hinchliffe hypothesized that three protons are indirectly pumped via three antiporter homologs, and the final proton is shifted in an unclear way. 

In addition, Tan et al speculated that the conformation changes and the density of water molecules in the trans-membrane domain determine the proton translocation in Complex I.

2. Complex II (CII)

Complex II (CII) namely, succinate dehydrogenase complex, is a component of the Krebs cycle as well as the ETC, serving as a link between metabolism and Oxphos. 

As a part of the Krebs cycle, Complex II catalyzes the oxidation of succinate to fumarate. 

Complex II is another entry point for electrons and donates them from succinate to CoQ via FeS clusters, similar to Complex I. 

Complex II consists of four subunits. A total of two of the subunits, the membrane-anchor proteins, are hydrophobic, anchor the complex to the inner membrane, and contain the CoQ binding site. 

The other two subunits are located on the matrix side of the inner membrane and contain the binding site of the substrate succinate, three FeS clusters and a flavoprotein covalently bound to a FAD cofactor. FAD is reduced to FADH₂ after receiving electrons from succinate and then transfers the electrons to FeS clusters. Then, CoQ is reduced to QH₂ after obtaining the electrons from the FeS cluster (3Fe-4S). Electron transport in Complex II is not accompanied by the translocation of protons. 

Succinate +UQ → Fumarate + UQH2

3. Complex III (CIII)

Complex III (CIII) is also referred to as a cytochrome bc_l complex, or CoQ-cytochrome c reductase and transfers the electrons carried by QH₂ to cytochrome c. Complex III is a symmetrical dimer with 11 subunits per monomer. 

The catalytically active subunits are cytochrome b (b_L to b_H), cytochrome c₁ and a high-potential (2Fe-2S) cluster wrapped by an iron-sulphur protein. There are two CoQ binding sites on both ends of cytochrome b embedded in the inner membrane of the mitochondria, one of which is the QH₂ oxidation site (Q_o) located at the cytoplasmic side, which is related to the low potential cytochrome b_L. The other is the Q^- reduction site (Q_i) on the side of the matrix, which is related to the high potential cytochrome b_H.

The electron transfer process of Complex III is accomplished by the Q-cycle. Ubiquinones, also known as CoEnzyme Q (CoQ), are lipid-soluble molecules found in the mitochondria of cells. They play a crucial role in the electron transport chain, a series of reactions essential for cellular energy production. 

Ubiquinones can exist in three oxidation states: fully oxidized (ubiquinone), partially reduced (semiquinone), and fully reduced (ubiquinol). These forms allow ubiquinones to function not only in electron transport but also as powerful antioxidants, protecting cells from oxidative damage.

QH₂ is oxidized to ubisemiquinone (QH^-) after transferring an electron to the (2Fe-2S) cluster and two protons are concurrently released into the mitochondrial intermembrane space (IMS) from the matrix. The (2Fe-2S) cluster transfers this electron to cytochrome c₁, from which it is transferred to cytochrome c, a mobile electron carrier. 

Then, the highly reductive QH⁻ formed at the Q_o site rapidly transfers the second electron to cytochrome b_L, which in turn transfers it to cytochrome b_H at the Q_i site. Reduced cytochrome b_H transfers this electron to the CoQ of the Q_i site, forming a QH⁻. 

To complete the Q-cycle, the second QH₂ molecule is oxidized at the Q_o site while displacing the other two protons. Similarly, one electron is transferred to the (2Fe-2S) cluster and the other electron to cytochrome b_H and finally to QH⁻ of the Qi site to produce QH₂.

UQH2+ 2 ferricytochrome C ⇆UQ +2 ferrocytochrome C + 2H +

4. Complex IV (CIV)

Complex IV (CIV) also known as cytochrome c oxidase, transfers electrons from cytochrome c to the terminal electron acceptor O₂ to generate H₂O. 

Mammalian Complex IV consists of 13 different subunits containing four redox-active metal centers, namely, Cu_A, heme a (Fe_a) and a binuclear center composed of heme a₃ (Fe_a3) and Cu_B. 

Subunits I, II, III are encoded by mitochondrial DNA and are the core subunits, while the 10 nuclear-coded subunits are the accessory subunits (encoded by nuclear DNA). 

Subunit I contains three of the four cofactors, heme a and the binuclear center, which transfers electrons from heme a to O₂. Subunits II and III are located on both sides of subunit I and there are two Cu_A cofactors on the side of the intermembrane space of subunit II. 

Subunit III stabilizes the other two core proteins and is mainly involved in proton pumping. The nuclear-coded subunits participate in the modulation of physiological activity via the allosteric ATP-mediated inhibition of Complex IV, which depends on the ATP/ADP-ratio.

Cytochrome c, similar to CoQ, is a mobile electron carrier that is loosely connected to the outer surface of the inner mitochondrial membrane by electrostatic interactions, allowing it to interact with the cytochrome c₁ of Complex III and to accept electrons . 

The reduced cytochrome c moves along the surface of the membrane and interacts with subunit II of Complex IV by electrostatic interactions, simultaneously transmitting electrons to the Cu_A site of subunit II, and then the electrons are passed from heme a to the binuclear center of subunit I, where the O₂ is reduced to H₂O. 

A total of four electrons at a time from cytochrome c are almost simultaneously transferred to bind dioxygen; eight protons in total are removed from the matrix, of which half are used to form the two water molecules and the other four are pumped across the membrane into the InterMembrane Space .

5. Complex V (CV)(ATP synthase ) and Mitochondrial oxidative phosphorylation

Complex V normally called F₁F₀ ATP synthase and consists of two functional domains: F₀ and F₁. The F₀ domain, located in the inner mitochondrial membrane, contains a subunit c-ring, including one of each of the subunits a, b, d, F6 and oligomycin sensitivity-conferring protein (OSCP) as well as the accessory subunits e, f, g and A6L. The subunits b, d, F6 and (oligomycin sensitivity-conferring protein)OSCP form the peripheral stalk, which is located on one side of the complex. 

A number of additional subunits (e, f, g and A6L), which all span the membrane, are associated with the c-ring subunit. The F₁ domain, situated in the mitochondrial matrix, consists of soluble subunits: Three α subunits, three β subunits and one of each of the γ, δ and ε subunits. 

The three α and three β subunits make up the catalytic head of F₁, and the γ, δ and ε subunits constitute the central stalk that connects the F₁ head and F₀ subunit c-ring. 

The ETC transfers two electrons at a time to monooxygen to generate one H₂O molecule, which is accompanied by the pumping of four, four and two protons from the matrix to the IMS through Complex I, Complex III, and Complex IV, respectively (or zero, four and two protons through the Complex II, Complex III, and Complex IV, respectively). 

Then, the protons pass from the IMS to the matrix through F₀, which transfers the stored energy created by the proton electrochemical gradient to F₁, causing a conformational change in F₁F₀ ATP synthase so that ADP can be phosphorylated to form ATP is called Oxphos(Oxidative Phosphorylation).

Due to the application of cryo-electron microscopy, a greater understanding of the high-resolution structure of these complexes has been gained (Oxidative phosphorylation).

2 ferrocytochrome –C+2H+ + 1/2O2 ⇆ 2 ferricytochrome-C +H2O

Two H-atoms or electrons from the reduced coenzyme (NADH or NADPH) travel through FAD and the cytochromes each with a more positive oxidation-reduction potential* and ultimately combine with ½ O₂ molecule to produce one molecule of H₂O. This is called Terminal oxidation.

The terminal oxidation of each reduced coenzyme requires ½ O₂, molecule and 2H atoms (i.e., 2e- +2H⁺) to produce one H₂O molecule. 

Except for flavoproteins (like FAD) and ubiquinone (UQ) which are hydrogen carriers, the other components of the electron transport chain (cytochromes) are only 'electron carriers' i.e They can not give or take protons (H⁺). 

Moreover, the above mentioned H-carriers form 'two electrons redox system' (i.e., they can give or take electrons at a time) while the cytochromes in electron transport chain form 'one electron redox systems' (i.e., they can donate or accept only one electron at a time). Therefore, to carry 2 electrons from the reduced coenzyme to 1⁄2 O₂ molecule, two molecules each of different cytochromes are needed. 

During this electron transport, FAD and the iron atom of different cytochromes get successively reduced (Fe⁺⁺) and oxidised (Fe⁺⁺⁺) and enough energy is released at some places which is utilised in the phosphorylation of ADP molecules (in the presence of inorganic phosphate) to generate energy rich ATP molecules.

3.0Sample Question on ETS and Oxidative Phosphorylation

Q.1 Given below is a diagram showing ATP synthesis during aerobic respiration, replace the symbols A, B, C, D and E by appropriate terms given in the box.

F1 Particle,Pi, 2H+, Inner mitochondrial membrane, ATP, Fo particle, ADP

Sol. A-ATP,B-F1particle,C-Pi,D-2H+,E-Inner mitochondrial space

4.0Multiple Choice Questions (MCQs)

Q.1 Match the following and choose the correct option from those given below. 

Column I Column II

A. Molecular oxygen i. α - Ketoglutaric acid 

B. Electron acceptor ii. hydrogen acceptor 

C. Pyruvate dehydrogenase iii. cytochrome C 

D. Decarboxylation iv. acetyl Co A 

Options are-

(1) A-ii, B-iii, C-iv, D-i (2) A-iii, B-iv, C-ii, D-i 

(3) A-ii, B-i, C-iii, D-iv (4) A-iv, B-iii, C-i, D-ii 

Ans. (1) A-ii, B-iii, C-iv, D-i

Q.2. The end product of oxidative phosphorylation is 

(1) NADH (2) Oxygen 

(3) ADP (4) ATP+H2O

Ans. (4) ATP+H2O

Q.3 Mitochondria are called powerhouses of the cell. Which of the following observations support this statement? 

(1) Mitochondria synthesise ATP 

(2) Mitochondria have a double membrane 

(3) The enzymes of the Krebs cycle are found in mitochondria. 

(4) Mitochondria are found in almost all plants and animal cells

Ans. (1) Mitochondria synthesise ATP 

Q.4. The ultimate electron acceptor of respiration in an aerobic organisms is: 

(1) Cytochrome (2) Oxygen

(3) Hydrogen (4) Glucose

Ans. (2) Oxygen

Q.5 Electron Transport System (ETS) is located in mitochondrial-

(1) Outer membrane (2) Inter membrane space 

(3) Inner membrane (4) Matrix

Ans. (3) Inner membrane