Biochemistry: A Short Course Second Edition

Tymoczko Berg Stryer Biochemistry: A Short Course Second Edition CHAPTERS 20 and 21 The Electron-Transport Chain And Oxidative Phosphorylation 2013 W. H. Freeman and Company Cellular Respiration and Physiologic Respiration Cellular respiration: the metabolic process by which an organism obtains energy by reacting oxygen with glucose to give water, carbon dioxide and ATP (energy). In physiology, respiration is defined as the transport of O 2 from the outside air to the cells within tissues, and the transport of CO 2 and H 2 O in the opposite direction. Physiologic respiration is necessary to sustain cellular respiration and thus life in animals. Cellular respiration CAC, Electron-transport chain and Oxidative Phosphorylation. The energy trapped in the form of high-energy electrons (CAC) is used for reduction of O 2 (electron-transport chain ) and the synthesis of ATP (oxidative phosphorylation). Whereas the CAC takes place in the mitochondrial matrix, electrontransport chain and the process of ATP synthesis take place in the mitochondrial inner membrane. 1

ATP Synthesis and Proton Gradients ATP need in humans: a sedentary male of 70 kg (154 lbs) requires ~ 85 kg of ATP/day. Humans possess only about 250 g of ATP, less than 1% of the daily required amount each ATP molecule is recycled from ADP approximately 300 times/day ATP recycling - primarily through oxidative phosphorylation-the culmination of cellular respiration Cellular respiration: high-transfer-potential electrons flow through a series of large protein complexes embedded in the inner mitochondrial membrane, called the respiratory chain, to reduce O 2 to H 2 O. The electron flow through these complexes is a series of highly exergonic oxidation reduction reactions that power the pumping of protons from the inside of the mitochondria to the outside, establishing a proton gradient, called the proton-motive force. The final phase of oxidative phosphorylation is carried out by an ATPsynthesizing assembly that is driven by the flow of protons back into the mitochondrial matrix shows that proton gradients are an interconvertible currency of free energy in biological systems. An overview of oxidative phosphorylation Oxidation and ATP synthesis are coupled by transmembrane proton fluxes. The respiratory chain (yellow structure) transfers electrons from NADH and FADH 2 to O 2 and simultaneously generates a proton gradient. ATP synthase (red structure) converts the energy of the proton gradient into ATP Respiration is an ATP-generating process in which an inorganic compound (such as molecular oxygen) serves as the ultimate electron acceptor. The electron donor can be either an organic compound or an inorganic one. Mitochondria Are Bounded by a Double Membrane The outer mitochondrial membrane is permeable to most small ions and molecules because of the channel protein mitochondrial porin. The inner membrane, which is folded into ridges called cristae, is impermeable to most molecules. The inner membrane is the site of electron transport and ATP synthesis. The citric acid cycle and fatty acid oxidation occur in the matrix. 2

Oxidative Phosphorylation Depends on Electron Transfer In the electron-transport chain, electrons from NADH and FADH 2 are used to reduce O 2 to H 2 O, in a highly exergonic reaction. The reduction of molecular oxygen by NADH and FADH 2 is accomplished through a number of electron-transfer reactions, which take place in a set of membrane proteins known as the electron-transport chain. Electrons from NADH and FADH 2 flow through the components of the electron chain. Figure 20.6 Components of the electron-transport chain The Electron Transfer Potential of an Electron Is Measured As Redox Potential In oxidative phosphorylation, the electron-transfer potential of NADH or FADH 2 is converted into the phosphoryl-transfer potential of ATP. The measure of phosphoryl-transfer potential is given by G for the hydrolysis of the phosphoryl compound. The corresponding measure for the electron transfer potential is E, the reduction potential (also called the redox potential or oxidation reduction potential). The reduction potential of a redox couple X/ X is measured experimentally by measuring the electromotive force generated by a sample halfcell connected to a standard reference half-cell Figure 20.4 The measurement of redox potential. Apparatus for the measurement of the standard oxidation reduction potential of a redox couple. Electrons flow through the wire connecting the cells, whereas ions flow through the agar bridge. The Redox Potential The sample half-cell consists of an electrode immersed in a solution of 1 M oxidant (X) and 1 M reductant (X ). The standard reference half-cell consists of an electrode immersed in a 1 M H + solution that is in equilibrium with H 2 gas at 1 atmosphere (1 atm). The electrodes are connected to a voltmeter, and an agar bridge allows the flow of ions between the half-cells. Electrons then flow from one half-cell to the other through the wire connecting the two cells. The reduction potential of the H + :H 2 couple is defined to be 0 volts. A negative reduction potential means that X has lower affinity for electrons than does H 2 ;it is a STRONGER REDUCING agent, and it has LOWER ELECTRON AFFINITY. The following reaction occurs: X + H + X ½ H Thus, a strong reducing agent (such as NADH) will donate electrons and has a negative reduction potential, whereas a strong oxidizing agent (such as O 2 ) will accept electrons and has a positive reduction potential. 3

Standard Redox Potential ΔE and standard free energy change ΔG The standard free-energy change G is related to the change in reduction potential E by G = nf E in which n is the number of electrons transferred, F is a proportionality constant called the faraday (96.48 kj mol 1 V 1, or 23.06 kcal mol 1 V 1 ), E is in volts, and G is in kilojoules or kilocalories per mole. For the reduction of molecular oxygen to water: This value is substantially negative indicating a highly spontaneous reaction The Electron Transport Chain Is a Series of Coupled Oxidation Reduction Reactions Electron flow from NADH to O 2 is accomplished by a series of intermediate electron carriers, that are coupled as members of sequential redox reactions. Electrons flow down the electron-transport chain until they finally reduce O 2. The members of the electron-transport chain are arranged so that the electrons always flow to components with a higher electron affinity. A more negative reduction potential means that X has lower affinity for electrons; more POSITIVE reduction potential means that X has HIGHER affinity for electrons Figure 20.10 The components of the electrontransport chain are arranged in complexes. The complexes shown in yellow boxes are proton pumps. Cyt c stands for cytochrome c. 4

Components of the electron transport chain Electrons flow down an energy gradient from NADH to O 2 through 4 large protein complexes embedded in the inner mitochondria membrane Fe and cytochrome c are component of all of the complexes. These complexes pump protons out of the mitochondria, generating a proton gradient. http://www.ncbi.nlm.nih.gov/ books/nbk21528/ Fe is a component of all of the complexes in the electron transport chain Figure 20.8 A heme component of cytochrome c oxidase. Fe is a component of all of the complexes in the electron transport chain For iron (Fe) to participate in several places in the electron-transport chain it must have several different reduction potentials. How can this happen? The oxidation reduction potential of iron ions can be altered by their environment. The iron ion is not free, it is embedded in different proteins, enabling iron to have various reduction potentials and to play a role at several different locations in the chain. In both iron sulfur proteins and cytochromes, iron shuttles between its oxidized ferric (Fe 3+ ) and its reduced ferrous state (Fe 2+ ). Figure 20.7 Iron sulfur clusters. (A) A single Fe ion bound by 4 cysteine residues. (B) 2Fe-2S cluster with iron ions bridged by sulfide ions. (C) 4Fe-4S cluster. Each of these clusters can undergo oxidation reduction reactions. 5

Coenzyme Q (CoQ) Another key feature of the electron-transport chain is the prominence of coenzyme Q (CoQ) as an electron carrier. Coenzyme Q, also known as ubiquinone because it is a ubiquitous quinone in biological systems, is a hydrophobic molecule which can diffuse rapidly within the inner mitochondrial membrane, where it shuttles protons and electrons. The most-common form in mammals contains 10 isoprene units (CoQ 10 ). Coenzyme Q (CoQ) Quinones can exist in three oxidation states (Figure 20.9). In the fully oxidized state (Q), coenzyme Q has two keto groups. The addition of one electron yields a semiquinone radical anion (Q ), whereas the addition of one electron and one proton results in the semiquinone form (QH ). The addition of a second electron and proton generates ubiquinol (QH 2 ), the fully reduced form of coenzyme Q. Thus, for quinones, electron-transfer reactions are coupled to proton binding and release, a property that is key to transmembrane proton transport. Respirasome The large protein complexes that transfer electrons in the respiratory chain are associated in a supramolecular complex termed the respirasome. As in the CAC, the complex facilitates the rapid transfer of substrate and prevent the release of reaction intermediates. The respirasome is composed of 4 large protein complexes: 1. NADH-Q oxidoreductase 2. Q cytochrome c oxidoreductase 3. Cytochrome c oxidase Electron flow within these transmembrane complexes leads to the transport of protons across the inner mitochondrial membrane. 4. Succinate-Q reductase, contains the succinate dehydrogenase that generates FADH 2 in the CAC. Electrons from this FADH 2 enter the electrontransport chain at Q-cytochrome c oxidoreductase. Succinate-Q reductase, in contrast with the other complexes, does not pump protons. 6

Components of the electron transport chain The electron transport chain High H + Low H + Figure 20.15 The electron-transport chain. High-energy electrons in the form of NADH and FADH 2 are generated by the citric acid cycle. These electrons flow through the respiratory chain, which powers proton pumping and results in the reduction of O 2. Electron Transport Chain And Oxidative Phosphorylation In the electron transport chain, electrons flow of from NADH to O 2, an exergonic process: NADH + ½O 2 + H + H 2 O + NAD + G = 220.1 kj/ mol During the electron flow, protons are pumped from the mitochondrial matrix to the outside of the inner mitochondrial membrane, creating a proton gradient. The energy of the proton gradient powers the synthesis of ATP (oxidative phosphorylation) ADP + P i + H + ATP G = +30.5 kj/ mol 7

A Proton Gradient Powers the Synthesis of ATP A molecular assembly ATP synthase, in the inner mitochondrial membrane, carries out the synthesis of ATP. In 1961, Peter Mitchell suggested the chemiosmotic hypothesis. He proposed that electron transport and ATP synthesis are coupled by a proton gradient across the inner mitochondrial membrane. The H + concentration becomes lower in the matrix membrane potential The ph gradient and membrane potential constitute a proton-motive force that is used to drive ATP synthesis Proton-motive force ( p) = chemical gradient ( H) + charge gradient ( Ψ) ATP Synthase Is Composed of a Proton Conducting Unit and a Catalytic Unit ATP synthase is a large, complex enzyme located in the inner mitochondrial membrane, composed of an F 0 and F 1 subunit : 1. F 0 subunit, is a hydrophobic segment that spans the inner mitochondrial membrane and contains the proton channel of the complex. This channel consists of a ring comprising from 8-15 c subunits, depending on the source of the enzyme, that are embedded in the membrane. A single a subunit binds to the outside of the ring. 2. F 1 subunit: 85-Å-diameter ball, protrudes into the mitochondrial matrix. Contains the catalytic activity of the synthase. Consists of 5 types of polypeptide chains (α 3, β 3, γ, δ, and ε). Figure 21.3 The structure of ATP synthase. A schematic structure of ATP synthase is shown. The F 0 subunit is embedded in the inner mitochondrial membrane, whereas the F 1 subunit resides in the matrix. ATP Synthase Mechanism: Rotational Catalysis http://www.youtube.com/watch?v=shs3lfu_ofm ATP synthase catalyzes the formation of ATP from ADP and orthophosphate. There are three active sites on the enzyme, each performing one of three different functions at any instant. The proton-motive force causes the three active sites to sequentially change functions as protons flow through the membrane-embedded component of the enzyme. The rotation of the γ subunit interconverts the three β subunits. Figure 21.4 ATP synthase nucleotide-binding sites are not equivalent. The γ subunit passes through the center of the α 3 β 3 hexamer and makes the nucleotidebinding sites in the β subunits distinct from one another. 8

An overview of oxidative phosphorylation Figure 21.10 An overview of oxidative phosphorylation. The electrontransport chain generates a proton gradient, which is used to synthesize ATP. If a resting human being requires 85 kg of ATP/day for bodily functions, then 3.3 10 25 protons must flow through ATP synthase/day, or 3.3 10 21 protons/second. Shuttles Allow Movement Across Mitochondrial Membranes The inner mitochondrial membrane must be impermeable to most molecules, yet much exchange has to take place between the cytoplasm and the mitochondria. This exchange is mediated by an array of membrane-spanning transporter proteins 9

Electrons from Cytoplasmic NADH Enter Mitochondria by Shuttles NADH cannot simply pass into mitochondria for oxidation by the respiratory chain, because the inner mitochondrial membrane is impermeable to NADH and NAD + electrons from NADH, rather than NADH itself, are carried across the mitochondrial membrane. Figure 21.11 The glycerol 3- phosphate shuttle. Electrons from NADH can enter the mitochondrial electron-transport chain by reducing DHAP to glycerol 3-phosphate. Electron transfer to an FAD prosthetic group in a membrane-bound glycerol 3- phosphate dehydrogenase reoxidizes glycerol 3- phosphate. Subsequent electron transfer to Q to form QH 2 allows these electrons to enter the electron-transport chain. The Entry of ADP into Mitochondria Is Coupled to the Exit of ATP ADP enters the mitochondrial matrix only if ATP exits, and vice versa. This process is carried out by the translocase, an antiporter: Figure 21.13 The mechanism of mitochondrial ATP-ADP translocase. The translocase catalyzes the coupled entry of ADP into the matrix and the exit of ATP from it. The binding of ADP (1) from the cytoplasm favors eversion of the transporter (2) to release ADP into the matrix (3). Subsequent binding of ATP from the matrix to the everted form (4) favors eversion back to the original conformation (5), releasing ATP into the cytoplasm (6) Mitochondrial Transporters Allow Metabolite Exchange Between the Cytoplasm and Mitochondria The inner mitochondrial membrane must be impermeable to most molecules, yet much exchange has to take place between the cytoplasm and the mitochondria. This exchange is mediated by an array of membrane-spanning transporter proteins. Figure 21.14 Mitochondrial transporters. Transporters (also called carriers) are transmembrane proteins that carry specific ions and charged metabolites across the inner mitochondrial membrane. 10

Cellular Respiration Is Regulated by the Need for ATP The stoichiometries of proton pumping and ATP synthesis, need not be integer numbers or even have fixed values. The best current estimates for the number of H + pumped out of the matrix by NADH-Q oxidoreductase, Q- cytochrome c oxidoreductase, and cytochrome c oxidase per electron pair are 4, 4, and 2, respectively (10 H + ) The synthesis of 1 molecule of ATP is driven by the flow of about 3 H + through ATP synthase. An additional H + is consumed in transporting ATP from the matrix to the cytoplasm Hence, about 2.5 molecules of cytoplasmic ATP are generated as a result of the flow of a pair of electrons from NADH to O 2. For electrons that enter at the level of Q- cytochrome c oxidoreductase,such as those from the oxidation of succinate or cytoplasmic NADH, the yield is about 1.5 molecules of ATP per electron pair. The Complete Oxidation of Glucose Yields About 30 Molecules of ATP The Rate of Oxidative Phosphorylation Is Determined by the Need for ATP Under most physiological conditions, electron transport is tightly coupled to phosphorylation. Electrons do not usually flow through the electron-transport chain to O 2 unless ADP is simultaneously phosphorylated to ATP. Experiments on isolated mitochondria demonstrate the importance of ADP level (Figure 21.15). The rate of oxygen consumption by mitochondria increases markedly when ADP is added and then returns to its initial value when the added ADP has been converted into ATP. When ADP concentration rises, as would be the case in active muscle that is continuously consuming ATP, the rate of oxidative phosphorylation increases to meet the ATP needs of the cell. The regulation of the rate of oxidative phosphorylation by the ADP level is called respiratory control or acceptor control 11

The energy charge regulates the use of fuels. Electrons do not flow from fuel molecules to O 2 unless ATP needs to be synthesized. The levels of ATP and ADP also affect the rate of the CAC - Figure 21.16 The energy charge regulates the use of fuels. The synthesis of ATP from ADP and P i controls the flow of electrons from NADH and FADH 2 to oxygen. The availability of NAD + and FAD in turn control the rate of the citric acid cycle (CAC). Regulated Uncoupling Leads to the Generation of Heat Some organisms possess the ability to uncouple oxidative phosphorylation from ATP synthesis to generate heat. In animals, brown fat (brown adipose tissue) is specialized tissue for this process of nonshivering thermogenesis. Brown adipose tissue is very rich in mitochondria, which contains a large amount of uncoupling protein 1 (UCP-1), also called thermogenin. UCP-1 forms a pathway for the flow of protons from the cytoplasm to the matrix. In essence, UCP-1 generates heat by short-circuiting the mitochondrial proton battery. New studies have established that adults, women especially, have brown adipose tissue on the neck and upper chest regions Figure 21.17 The action of an uncoupling protein. Uncoupling protein 1 (UCP-1) generates heat by permitting the influx of protons into the mitochondria without the synthesis of ATP. The energy is used for generating heat. Oxidative Phosphorylation Can Be Inhibited at Many Stages Many potent and lethal poisons exert their effects by inhibiting oxidative phosphorylation at one of a number of different locations. Inhibition of the electron-transport chain: rotenone, which is used as a fish and insect poison; amytal, a barbiturate sedative; antimycin A, an antibiotic isolated from Streptomyces that is used as a fish poison; cyanide (CN ); azide (N 3 ); and carbon monoxide (CO). Inhibition of ATP synthase : Oligomycin, an antibiotic used as an antifungal agent, and dicyclohexylcarbodiimide (DCCD), used in peptide synthesis in the laboratory,) Uncoupling electron transport from ATP synthesis: 2,4- dinitrophenol (DNP) and certain other acidic aromatic compounds Inhibition of ATP export: atractyloside (a plant glycoside) or bongkrekic acid (an antibiotic from a mold) 12

Inhibition of the electron transport chain: Figure 21.20 Sites of action of some inhibitors of electron transport. The proton gradient is an interconvertible form of free energy Proton gradients power a variety of energy-requiring processes such as: the active transport of calcium ions by mitochondria, the entry of some amino acids and sugars into bacteria, the rotation of bacterial flagella, and the transfer of electrons from NADP + to NADPH. As we have already seen, proton gradients can also be used to generate heat 13