15.1. Mitochondrial Structure
A mitochondrion contains two membranes: an outer membrane, which is responsible for maintaining the shape of the organelle, and a much less permeable inner membrane. The outer membrane contains porin, creating a "molecular sieve. a protein that forms pores large enough allow molecules less than ~10 kDa to diffuse freely across the membrane.
The region between the membranes is called the intermembrane space. The inner membrane is largely impermeable for molecules and acts as a barrier to prevent the movement of most molecules. The inner membrane contains cristae, which are involutions in inner membrane. The function of the cristae is to increase the surface area than the cell plasma membrane, due to the involutions in the membrane.
The inner and outer membranes also differ in their composition of lipids and proteins. Cardiolipin, a major phospholipid component of the inner membrane, is absent from the outer membrane. Phosphatidylinositol and cholesterol are important constituents of the outer membrane, but are absent from the inner membrane.
The outer mitochondrial membrane is involved in phospholipid biosynthesis as it contains a number of enzymes. No proteins in the outer membrane are coded by any genes in the mitochondrial genome. A few molecules have specific transporters that allow them to enter or exit the mitochondrion.
The TCA cycle enzymes are located in the matrix. TCA cycle enzymes (including succinate dehydrogenase) are all produced from nuclear genes; the multisubunit complexes of the electron transport pathway and ATP synthase (with the exception of succinate dehydrogenase) are made up of proteins derived from both nuclear and mitochondrial genes.
15.2. The Components of the Electron Transport Chain
The electron transport chain of the mitochondria is the means by which electrons are removed from the reduced carrier NADH and transferred to oxygen to yield H2O.
NADH is generated in the matrix by the reactions of pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehyrogenase. The electron transport chain begins with reoxidizing NADH to form NAD+ and channeling the electrons into the formation of reduced coenzymes. Important to note that NADH transfers 2 electrons at a time in the form of a hydride.
Flavoproteins have either a FAD (flavin adenosine dinucleotide) or a FMN (flavin mononucleotide) as prosthetic group. Flavoproteins can accept or donate electrons one at time or two at a time. Thus they are often intermediaries between two electron acceptors/donors and one electron acceptors/donors. For flavoproteins the typical standard reduction potentials are around 0 V.
15.2.3. Coenzyme Q (CoQ) Or ubiquinone (UQ).
CoQ is made up of ten repeating isoprene units. It is soluble in the hydrophobic lipid bylayer. Coenzyme Q is a soluble electron carrier present in the hydrophobic lipid bilayer of the inner mitochondrial membrane. Like flavoproteins, CoQ can accept/donate electrons one at a time or two at a time.
Ubiquinone (UQ) is ubiquitous nature, as it occurs virtually in all cells. It was formerly called as coenzyme Q (Q for quinone) and abbreviated as CoQ or simply Q.
Ubiquinone is actually a group of compounds, all containing the same quinone structure but substituted with a long side chain composed of varying numbers (from 6 to 10) of isoprene units (=prenyl groups), linked head to tail. The isoprenoid tail makes ubiquinone highly nonpolar which enables it to diffuse readily in the hydrocarbon phase of the inner mitochondrial membrane. Ubiquinone is the only electron carrier in the respiratory chain that is not tightly bound or covalently attached to a protein. In fact, it serves as a highly mobile carrier of electrons between the flavoproteins and the cytochromes of the electron-transport chain.
The closely related plastoquinones, which function as an analogous carrier of electrons in photosynthesis, differ from ubiquinones in the alkyl substituents of the benzene ring : two –CH3 groups instead of two –OCH3 and H instead of –CH3. Plastoquinones B and C carry one hydroxyl group in the side chain.
Cytochromes are proteins that contain heme prosthetic groups which function as one electron carriers. They are found either as monomeric proteins (e.g., cytochrome c) or as subunits of larger enzymatic complexes that catalyze redox reactions.
The heme iron is involved in one electron transfers involving the Fe2+ and Fe3+ oxidation states. Thus cytochromes are capable of performing oxidation and reduction. Because the cytochromes (as well as other complexes) are held within membranes in an organized way, the redox reactions are carried out in the proper sequence for maximum efficiency.
Cytochromes enzyme complex catalyzing the oxidation of ubiquinol (QH2) contains an iron-sulfide protein of Fe2S2 Cys4 type and 2 types of cytochromes. The iron atom in cytochromes alternates between a reduced ferrous (Fe2+) state and an oxidized ferric (Fe3+) state during electron transport. A heme group, like an Fe –S centre, is one-electron carrier, in contrast with NADH, flavins and ubiquinone, which are two electron carriers.
Five types of cytochromes are present between ubiquinol (QH2) and oxygen in the electron-transport chain. Based upon light-absorption spectra they are classified as a, b, c and so on. cytochrome a absorbing at the longest wavelength called that absorbing the next longest wavelength called cytochrome b, and so on.
Unfortunately, the order of wavelength does not correspond to the physiological sequence in which they function :
b c1 c a a3
The prosthetic group of cytochromes b, c and c1 is iron-protoporphyrin IX, commonly called heme or hemin. Heme prosthetic group is also present in myoglobin, hemoglobin, catalase and peroxidase. In cytochromes c and c1, the heme is covalently attached to the protein by thioether linkages but not in the other cytochrome.
The type of heme present in cytochrome b is called heme B and the one present in cytochromes c and c1 as heme C.
Three types of cytochrome are distinguished by their prosthetic groups:
Type prosthetic group
Cytochrome a heme a
Cytochrome b heme b
Cytochrome d tetrapyrrolic chelate of iron
In mitochondria and chloroplasts, these cytochromes are often combined in electron transport and related metabolic pathways:
A completely distinct family of cytochromes is known as the cytochrome P450 oxidases, cytochrome P450 oxidases absorbance light at wavelengths 450 nm. These enzymes are primarily involved in steroidogenesis and detoxification.
The cytochromes a and a3 have heme A.
Cytochromes a and a3 are the terminal members of the respiratory chain. They exist as complex, which is sometimes called cytochrome oxidase. Cytochrome c is the only electron-transport protein that can be separated from the inner mitochondrial membrane by gentle treatment. The hydrophobic nature of the heme of cytochrome c makes the redox potential of cytochrome c more positive, corresponding to a higher electron affinity.
15.2.5. Iron-Sulphur Proteins
In the electron transport chain we will encounter many iron-sulphur proteins which participate in one electron transfers involving the the Fe2+ and Fe3+ oxidation states. These are non-heme iron-sulphur proteins. The simplest iron-sulfer protein is FeS in which iron is tetrahedrally coordinated by four cysteines. The second form is Fe2S2 which contains two irons complexed to 2 cysteine residues and two inorganic sulfides.
The third form is Fe3S4 which contains 3 iron atoms coordinated to three cysteine residues and 4 inorganic sulfides. The last form is the most complicated Fe4S4 which contains 4 iron atoms coordinated to 4 cysteine residues and 4 inorganic sulfides.
The iron atoms in these complexes can be in the reduced (Fe2+) or oxidized (Fe3+) state. An important feature of the iron-sulfur proteins is that their relative affinity for electrons can be varied over a wide range by changing the nature of the polypeptide chain. Some are relatively strong oxidizing agents ; others are powerful reducing agents — even stronger than NADH. NADH dehydrogenase contains both the Fe2S2Cys4 and Fe4S4Cys4 types of complexes.
15.2.6 . Copper Proteins
Copper bound proteins participate in one electron transfers involving the Cu+ and Cu2+ oxidation states.
15.3. Overview of the Electron Transport Chain
Electrons move along the electron transport chain going from donor to acceptor until they reach oxygen the ultimate electron acceptor. The standard reduction potentials of the electron carriers are between the NADH/NAD+ couple (-0.315 V) and the oxygen/H2O couple (0.816 V) Following four enzymes works in the electron transport chain –
I NADH-CoQ Oxidoreductase
II FADH2-CoQ Oxidoreductase
III CoQ-Cyt C Oxidoreductase
IV Cyt C Oxidase
One another enzyme ATP Synthase forms complex V which is used for ATP synthesis from Proton motive force. ATP synthase is the membrane embedded enzyme which is made up of several subunits.
Out of 4 enzymes complex II is not able to generate PMF hence it is not able to transport H+ ions across the membrane when it transferes its electrons from FADH2 to COQ. The movement of electrons is dependent upon the standard reduction potential of any molecule which means the ability to donate or accept the electrons of any molecule. Electrons move from the lowest reduction potential to highest reduction potential. Oxygen has the highest (most positive) standard reduction potential which means that it have the highest acceptance of electrons from other carriers. The order of the individual electron carriers in the chain was determined by spectroscopy.
15.3.1. Complex I (NADH-coenzyme Q reductase)
Complex I accepts electrons from NADH and serves as the link between glycolysis, the citric acid cycle, fatty acid oxidation and the electron transport chain. The NADH dehydrogenase complex is the largest of the respiratory enzyme complexes, which contains more than 40 polypeptide chains. Complex I catalyses oxidation of NADH by CoQ(ox). It accepts electrons from NADH and passes them through a flavin and iron-sulphur centers to ubiquinone. Ubiquinone then transfers its electrons to a second respiratory enzyme complex known as the cytochrome b-c1 complex.
This enzyme is consists of Flavin mononucleotide (FMN) and Iron-sulfur proteins. FMN differs from NAD only by absence of AMP group. The Iron-sulfur proteins are second major family of electron carriers. These are arranged as 8 or 9 cluster in either the 2Fe-2S type or the 4Fe-4S type iron-sulfur center and bound to cysteine side chains on the protein. The prosthetic group FMN is absolutely required for activity. Therefore this complex is a flavoprotein. These compounds are detected by electron spin resonance (ESR) spectroscopy.
This complex binds NADH, transfers two electrons in the form of a hydride to FMN to produce NAD+ and FMNH2. The subsequent steps involve the transfer of electrons once at a time to a series of iron-sulphur complexes that includes both 2Fe-2S and 4Fe-4S clusters.
Importance of FMN
It functions as a 2 electron acceptor in the hybrid transfer from NADH. Second it functions as a 1 electron donor to the series of iron sulphur clusters. FMN and FAD often play crucial links between 2 electron transfer agents and 1 electron transfer agents. The final step of this complex is the transfer of 2 electrons once at a time to coenzyme Q. CoQ like FMN and FAD can function as a 2 electron donor/acceptor and as a 1 electron donor/acceptor. CoQ is a mobile electron carrier because its isoprenoid tail makes it highly hydrophobic and lipophilic. It diffuses freely in the bilipid layer of the inner mitochondrial membrane.
The intermembrane space side of the inner membrane is referred to as the P face (P standing for positive). The matrix side of the inner membrane is referred to as the N face. The transport of electrons from NADH to CoQ is coupled to the transport of protons across the membrane. This is an example of active transport. The stoichiometry is 4 H+ transported per 2 electrons.
15.3.2. Complex II
This enzyme complex is named as FADH2- CoQ oxidoreductase which is also termed as succinate dehydrogenase complex. It is a membrane bound enzyme that also participates in Krebs Cycle, oxidizing succinate into malate and releasing FADH2. This FADH2 is transferred to different carriers to the CoQ. Complexes I and II both produce reduced coenzyme Q,CoQH2 which is the substrate for Complex III.
The only enzyme of the citric acid cycle that is an integral membrane protein. This complex is composed of four subunits. 2 of which are iron-sulfur proteins and the other two subunits together bind FAD through a covalent link to a histidine residue. These two subunits are called flavoprotein 2 or FP2. Complex II contains three Fe-S centers, one 4Fe-4S cluster, one 3Fe-4S cluster and one 2Fe- 2S cluster. In the first step of this complex, succinate is bound and a hydride is transferred to FAD to generate FADH2 and fumarate. FADH2 then transfers its electrons one at a time to the Fe-S centers. Thus once again FAD functions as 2 electron acceptor and a 1 electron donor. The final step of this complex is the transfer of 2 electrons one at a time to coenzyme Q to produce CoQH2.
For complex II the standard free energy change of the overall reaction is too small to drive the transport of protons across the inner mitochondrial membrane. This accounts for the 1.5 ATP’s generated per FADH2 compared with the 2.5 ATP’s generated per NADH.
15.3.3. Complex III
This complex is also known as coenzyme Q - cytochrome c reductase because it passes the electrons form CoQH2 to cyt c through a very unique electron transport pathway called the Q-cycle.
Ubiquinone(Q) or coenzyme Q are the simplest electron carriers in the respiratory chain which are hydrophobic in nature and are nor associated with protein and freely mobile in the lipid bilayer. It can pick up or donate either one or two electrons and upon reduction, it picks up a proton with each electron it carries.
The cytochrome b-c1 complex contains at least 11 different polypeptide chains and functions as a dimer. Each monomer unit of cytochrome b-c1 complex contains three hemes bound to cytochrome and an iron-sulphur protein. The complex accepts electrons from ubiquinone and passes them on to cytochrome c, which transfers its electron to the cytochrome oxidase complex.
The Q-cycle is initiated when CoQH2 diffuses through the bilipid layer to the CoQH2 binding site which is near the intermembrane face. This CoQH2 binding site is called the QP site. The electron transfer occurs in two steps. First one electron from CoQH2 is transferred to the Rieske protein (a Fe-S protein) which transfers the electron to cytochrome c1. This process releases 2 protons to the intermembrane space.
(a) First half of Q cycle
Coenzyme Q is now in a semiquinone anionic state, CoQHx - still bound to the QP site. The second electron is transferred to the bL heme which converts CoQHx - to CoQ. This reoxidized CoQ can now diffuse away from the QP binding site. The bL heme is near the P-face. The bL heme transfers its electron to the bH heme which is near the N-face. This electron is then transferred to second molecule of CoQ bound at a second CoQ binding site which is near the N-face and is called the QN binding site. This electron transfer generates a CoQx - radical which remains firmly bound to the QN binding site. This completes the first half of the Q cycle.
(b) Second half of Q cycle
The second half of the Q-cycle is similar to the first half. A second molecule of CoQH2 binds to the QP site. In the next step, one electron from CoQH2 (bound at QP) is transferred to the Rieske protein which transfers it to cytochrome c1. This process releases another 2 protons to the intermembrane space. The second electron is transferred to the bL heme to generate a second molecule of reoxidized CoQ. The bL heme transfers its electron to the bH heme. This electron is then transferred to the CoQ-radical still firmly bound to the QN binding site. The take up of two protons from the N-face produces CoQH2 which diffuses from the QN binding site. This completes Q cycle.
The net result of the Q-cycle is 2e- transported to cytochrome c1. Two protons were picked up from the N-face in the second half of the Q-cycle and 4 protons total were released into the intermembrane space. The two electron carrier CoQH2 gives up its electrons one at a time to the Rieske protein and the bL heme both of which are one electron carriers.
The electrons that end up on cytochrome c1 are transferred to cytochrome c. Cytochrome c is the only water soluble cytochrome. Cytochrome c is coordinated to ligands that protect the iron contained in the heme from oxygen and other oxidizing agents. Cytochrome c is a mobile electron carrier that diffuses through the intermembrane space shuttling electrons from the c1 heme of complex III to CuA site of complex IV.
Cytochrome c shown to the left. The heme is linked to the protein by 4 cysteine linkages shown in yellow. A methionine sulfur atom is coordinated to the iron compexed in the heme. A histidine residue protects the iron from oxygen and other potential ligands.
15.3.5. Complex IV
The cytochrome oxidase complex also functions as a dimer. Each monomer contains 13 different polypeptide chains, including two cytochromes and two copper atoms. The complex accepts one electron at a time from cytochrome c and passes them to oxygen. The oxidation state of Fe+2 is converted to Fe+3 upon the addition of electron.
Oxygen has a high affinity for electrons so it releases a large amount of free energy when it is reduced to form water. This is the evolution of cellular respiration and which enabled organisms to harness much more energy compared to anaerobic respiration.
The cell is able to use O2 for respiration because of the cytochrome oxidase which holds oxygen between a heme-linked iron atom and a copper atom until it gets all its four electrons and released as 2H2O.
Cytochrome c oxidase contains 2 heme centers, cytochrome a and cytochrome a3 and two copper proteins. The copper sites are called CuA and CuB. CuA is associated with cytochrome a and CuB is associated with cytochrome a3 .
The copper sites function as one electron carriers cycling between the cuprous state Cu+ and the cupric state Cu2+ Just like iron sulphur proteins. Cytochrome a transfers one electron to CuB.
A second cytochrome c binds and transfer its electron to CuA. CuA subsequently transferred its electron to cytochrome a which in turn is transferred to cytochrome a3. Cytochrome c is bound on the P-face of the Inner mitochondrial membrane and transfers its electron to CuA. The oxidized cytochrome c get dissociates from complex 4. CuA then transfers the electron to cytochrome a. Cytochrome a transfers the electron to CuB. A second cytochrome c binds and transfer its electron to CuA which is subsequently transferred to cytochrome a which in turn is transferred to cytochrome a3. The binuclear metal center now has two electrons bound allowing the binding of O2 to binuclear center. The next step involves the uptake of two protons and the transfer of yet another electron through the same pathway which leads to cleavage of the O-O bond and the generation of a Fe4+ metal center. The fourth electron is transferred to form a hydroxide at the heme center which becomes protonated and dissociates as H2O. The mechanism is shown below. The reduction of oxygen by complex IV involves the transfer of four electrons. Four protons are abstracted from the matrix and two protons are released into the intermembrane space. The reduction of oxygen by complex IV involves the transfer of four electrons. Four protons are abstracted from the matrix and two protons are released into the intermembrane space.
15.3.6. The mitochondrial electron-transport chain-
The standard reduction potentials of its most mobile components are indicated, as are the points where sufficient free energy is harvested to synthesize ATP and the sites of action of several respiratory inhibitors. The Complexes I, III, and IV do not directly synthesize ATP but, rather, sequester the free energy necessary to do so by pumping protons outside the mitochondrion to form a proton gradient.
15.4. Inhibitor of ETC
There are many inhibitor molecules which reveals the working of electron-transport chain. Following are some compounds –
Effect of inhibitors on electron transport. This diagram shows an idealized oxygen electrode trace of a mitochondrial suspension containing excess ADP and Pi. At the numbered points, the indicated reagents are injected into the sample chamber and the resulting changes in [O2] are recorded.
In ETS system, as we added -hydroxy-butyrate, NAD+ linked oxidation commences as -hydroxy butyrate is the source of ketone bodies. During starvation, its production increases.
As we add rotenone or amytal it interfere on inhibit NAD+ linked oxidation as it inhibit the transfer of electrons from iron-sulphur enters in complex 1 to ubiquinone. As cellular oxygen gets reduced so it creates ROS (relative oxygen species which can damage DNA and components of mitochondria).
In ETS system as succinate is added so it is generated in mitochondria by tricarboxylic acid cycle (TCA), succinate can exit the mitochondrial matrix only functional in cytoplasm as well as extra cellular space changing gene expression.
As succinate added, FAD linked glycosylation begins but antimycin is added it inhibits FAD-linked oxidation.
Cyanide is an inhibitor of enzyme cytochrome oxidase in the IV complex of ETS; as it prevent O2 from binding. As of now, O2 cannot bind nor can reduced to water. Because of this, mitochondria can't make ATP.