Chapter 20 - Oxidative Phosphorylation PDF

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General Biochemistry
Instructor - Sara Casado Zapico...

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The study of oxidative phosphorylation begins by examining the oxidation–reduction reactions that allow the flow of electrons from NADH and FADH to oxygen. The electron flow, which is very exergonic, takes place in four large protein complexes that are embedded in the inner mitochondrial membrane, together called the respiratory chain or the electron- transport chain. Importantly, three of these complexes use the energy released by the electron flow to pump protons from the mitochondrial matrix into the space between the inner and outer mitochondrial membranes. The proton gradient is then used to power the synthesis of ATP by oxidative phosphorylation. Collectively, the generation of high-transfer-potential electrons by the citric acid cycle, their flow through the respiratory chain, and the accompanying synthesis of ATP is called respiration or cellular respiration.

20.1 Oxidative Phosphorylation in Eukaryotes Takes Place in Mitochondria Like the citric acid cycle, the respiratory chain and ATP synthesis take place in mitochondria. Whereas the citric acid cycle takes place in the mitochondrial matrix, the flow of electrons through the respiratory chain and the process of ATP synthesis take place in the mitochondrial inner membrane. Mitochondria Are Bounded by a Double Membrane

Electron microscopic studies reveal that mitochondria have two membrane systems: an outer membrane and an extensive, highly folded inner membrane. The inner membrane is folded into a series of internal ridges called cristae. Hence, there are two compartments in mitochondria: (1) the intermembrane space between the outer and the inner membranes and (2) the matrix, which is bounded by the inner membrane. The mitochondrial matrix is the site of the reactions of the citric acid cycle and fatty acid oxidation. In contrast, oxidative phosphorylation takes place in the inner mitochondrial membrane. The increase in surface area of the inner mitochondrial membrane provided by the cristae creates more sites for oxidative phosphorylation. The outer membrane is quite permeable to most small molecules and ions because it contains many copies of mitochondrial porin, a poreforming protein also known as VDAC, for voltage-dependent anion channel. VDAC regulates the flux of molecules crucial to the function of cellular respiration—usually anionic species such as phosphate, components of the citric acid cycle, and the adenine nucleotides—across the outer membrane. In contrast, the inner membrane is intrinsically impermeable to nearly all ions and polar molecules. A large family of transporters shuttle metabolites such as ATP, pyruvate, and citrate across the inner mitochondrial membrane. The two faces of this membrane will be referred to as the matrix side and the cytoplasmic side (the latter because it is freely accessible to most small molecules in the cytoplasm). In bacteria, the electron-driven proton pumps and ATPsynthesizing complex are located in the plasma membrane.

20.2 Oxidative Phosphorylation Depends on Electron Transfer The primary catabolic function of the citric acid cycle is the generation of NADH and FADH 2 by the oxidation of acetyl CoA. In oxidative phosphorylation, electrons from NADH and FADH2 are used to reduce molecular oxygen to water. The highly exergonic reduction of molecular oxygen by NADH and FADH2 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 FADH2 flow through the components of the electron chain, ultimately resulting in the reduction of oxygen. The Electron-Transfer Potential of an Electron Is Measured as Redox Potential

In oxidative phosphorylation, the electron-transfer potential of NADH or FADH2 is converted into the phosphoryltransfer potential of ATP. To better understand this conversion, we need quantitative expressions for these forms of free energy. The measure of phosphoryl-transfer potential is already familiar to us: it is given by ∆G°′ for the hydrolysis of the phosphoryl compound. The corresponding expression for the electron-transfer potential is E′0, the reduction potential (also called the redox potential or oxidation–reduction potential). A negative reduction potential means that the oxidized form of a substance has lower affinity for electrons than does H2, as in the preceding example. A positive reduction potential means that the oxidized form of a substance has higher affinity for electrons than does H2. These comparisons refer to standard conditions—namely, 1 M oxidant, 1 M reductant, 1 M H+, and 1 atm H2. Thus, a strong reducing agent (such as NADH) is poised to donate electrons and has a negative reduction potential, whereas a strong oxidizing agent (such as O2) is ready to accept electrons and has a positive reduction potential. Electron Flow Through the Electron-Transport Chain Creates a Proton Gradient

The driving force of oxidative phosphorylation is the electron-transfer potential of NADH or FADH2 relative to that of O2. Recall that NADH and FADH2 are carriers of high-transfer-potential electrons generated in the citric acid cycle and else- where in metabolism. The energy released by the reduction of each electron carrier generates a proton gradient that is then used for the synthesis of ATP and the transport of metabolites across the mitochondrial membrane. The Electron-Transport Chain Is a Series of Coupled Oxidation–Reduction Reactions

Electron flow from NADH to O2 is accomplished by a series of intermediate electron carriers—a bucket brigade of electron carriers—that are coupled as members of sequential redox reactions. NADH is oxidized by passing electrons to flavin mononucleotide (FMN), an electron carrier similar to flavin adenine dinucleotide (FAD) but lacking the nucleotide component. Reduced FMN is subsequently oxidized by the next electron carrier in the chain, and the process repeats itself as the electrons flow down the electron-transport chain until they finally reduce O2. The members of the electron-transport chain are arranged so that the electrons always flow to components with more positive reduction potentials (a higher electron affinity). Electrons are transferred from NADH to O2 through a chain of three large protein complexes called NADH- Q oxidoreductase, Q-cytochrome c oxidoreductase, and cytochrome c oxidase. These complexes appear to be associated in a supramolecular complex termed the respirasome. As in the citric acid cycle, such supramolecular complexes facilitate the rapid transfer of substrate and prevent the release of reaction intermediates. A fourth large protein complex, called succinate-Q reductase, contains the succinate dehydrogenase that generates FADH2 in the citric acid cycle. Electrons from this FADH2 enter the electron-transport chain at Q-cytochrome c oxidoreductase.

Electrons from FADH2 feed into the chain “downstream” of those from NADH because the electrons of FADH 2 have a lower reduction potential. As a result, FADH2-derived electrons pump fewer protons and thus yield fewer molecules of ATP. Second, note that iron is a prominent electron carrier, appearing in several places. Iron in the electron-transport chain appears in two fundamental forms: associated with sulfur as iron–sulfur clusters located in iron–sulfur proteins (also called nonheme-iron proteins), and as components of a heme-prosthetic group, which are embedded in a special class of proteins called cytochromes. In both iron–sulfur proteins and cytochromes, iron shuttles between its reduced ferrous (Fe2+ ) and its oxidized ferric state (Fe3+ ):

The heme-prosthetic group of cytochromes is iron-protoporphyrin IX, the same heme present in hemoglobin and myoglobin. The iron in hemoglobin and myoglobin, in contrast to the iron in cytochromes, remains in the Fe2+ oxidation state. The fact that iron is an electron carrier in several places in the electron-transport chain raises a puzzling question. If the flow of electrons from NADH to O2 is exergonic and iron has a reduction potential of +0.77 V, how can iron participate in several places in the electron-transport chain if each step is exergonic? In other words, how can iron have several different reduction potentials? The answer to the puzzle is that the oxidation –reduction potential of iron ions can be altered by their environment. In regard to the electron-transport chain, the iron ion is not free; rather, 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. The metal copper also appears as a member of the final component of the electron-transport chain, where it is alternatively oxidized and reduced: Another key feature of the electron-transport chain is the prominence of coenzyme Q (Q) as an electron carrier. Coenzyme Q, also known as ubiquinone because it is a ubiquitous quinone in biological systems, is a quinone

derivative with a long isoprenoid tail, which renders the molecule hydrophobic and allows it to diffuse rapidly within the inner mitochondrial membrane, where it shuttles protons and electrons about. Coenzyme Q consists of more than one five-carbon isoprene unit. The exact number depends on the species in which it is found. The most common form in mammals contains 10 isoprene units (coenzyme Q 10). For simplicity, the subscript will be omitted from this abbreviation because all varieties function in an identical manner. Quinones can exist in several oxidation states.

20.3 The Respiratory Chain Consists of Proton Pumps and a Physical Link to the Citric Acid Cycle Electron flow within these transmembrane complexes leads to the transport of protons across the inner mitochondrial membrane. A fourth protein complex, succinate-Q reductase, in contrast with the other complexes, does not pump protons. The High-Potential Electrons of NADH Enter the Respiratory Chain at NADH-Q Oxidoreductase

The electrons of NADH enter the chain at NADH-Q oxidoreductase (also called Complex I and NADH dehydrogenase), an enormous enzyme consisting of approximately 46 polypeptide chains and two types of prosthetic groups: FMN and iron –sulfur clusters. This proton pump is L-shaped, with a hydrophobic horizontal arm lying in the membrane and a hydrophilic vertical arm that projects into the matrix. The electrons flow from NADH to FMN and then through a series of seven iron –sulfur clusters to Q. Note that all of the redox reactions take place in the extramembranous part of NADH-Q oxidoreductase. Although the precise stoichiometry of the reaction catalyzed by this enzyme is not completely worked out, it appears to be

Recent structural studies have suggested how Complex I acts as a proton pump. What are the structural elements required for proton pumping? The membrane-embedded part of the complex has four proton half-channels consisting, in part, of vertical helices. One set of

half- channels is exposed to the matrix and the other to the intermembrane space. The vertical helices are linked on the matrix side by a long horizontal helix (HL) that connects the matrix half-channels, while the intermembrane space half-channels are joined by a series of b-hairpin-helix connecting elements (bH). An enclosed Q chamber, the site where Q accepts electrons from NADH, exists near the junction of the hydrophilic portion and the membraneembedded portion. Finally, a hydrophilic funnel connects the Q chamber to a water-lined channel, into which the half-channels open, that extends the entire length of the membrane-embedded portion. How do these structural elements cooperate to pump protons out of the matrix? When Q accepts two electrons from NADH, generating Q2−, the negative charges on Q2− interact electrostatically with negatively charged amino acid residues in the membrane-embedded arm, causing conformational changes in the long horizontal helix and the bH elements. These changes in turn alter the structures of the connected vertical helices that change the pKa of amino acids, allowing protons from the matrix to first bind to the amino acids, then dissociate into the waterlined channel and finally enter the intermembrane space. Thus, the flow of two electrons from NADH to coenzyme Q through NADH-Q oxidoreductase leads to the pumping of four hydrogen ions out of the matrix of the mitochondrion. Q2− subsequently takes up two protons from the matrix as it is reduced to QH2. The removal of these protons from the matrix contributes to the formation of the proton-motive force. The QH subsequently leaves the enzyme for the Q pool, allowing another reaction cycle to occur. Ubiquinol Is the Entry Point for Electrons from FADH2 of Flavoproteins

Recall that FADH2 is formed in the citric acid cycle in the oxidation of succinate to fumarate by succinate dehydrogenase. This enzyme is part of the succinate-Q reductase complex (Complex II), an integral membrane protein of the inner mitochondrial membrane. The electron carriers in this complex are FAD, iron –sulfur proteins, and Q. FADH2 does not leave the complex. Rather, its electrons are transferred to Fe-S centers and then to Q for entry into the electron-transport chain. The succinate-Q reductase complex, in contrast with NADH- Q oxidoreductase, does not transport protons. Consequently, less ATP is formed from the oxidation of FADH 2 than from NADH. Electrons Flow from Ubiquinol to Cytochrome c Through Q-Cytochrome c Oxidoreductase

The second of the three proton pumps in the respiratory chain is Q-cytochrome c oxidoreductase (also known as Complex III and cytochrome c reductase). The function of Q-cytochrome c oxidoreductase is to catalyze the transfer of electrons from QH2 produced by NADH-Q oxidoreductase and the succinate-Q reductase complex to oxidized cytochrome c (Cyt c), a water-soluble protein, and concomitantly pump protons out of the mitochondrial matrix. The flow of a pair of electrons through this complex leads to the effective net transport of 2 H+ to the intermembrane space, half the yield obtained with NADH-Q oxidoreductase because of a smaller thermodynamic driving force.

The Q Cycle Funnels Electrons from a Two-Electron Carrier to a One-Electron Carrier and Pumps Protons

QH2 passes two electrons to Q-cytochrome c oxidoreductase, but the acceptor of electrons in this complex, cytochrome c, can accept only one electron. How does the switch from the two-electron carrier ubiquinol to the one-electron carrier cytochrome c take place? The mechanism for the coupling of electron transfer from Q to cytochrome c to transmembrane proton transport is known as the Q cycle. The cycle begins when two QH2 molecules bind to the complex consecutively, each giving up two electrons and two H+. These protons are released to the intermembrane space. QH2 binds to the first Q binding site (Qo), and the two electrons travel through the complex to different destinations. One electron flows, first, to the Rieske 2Fe-2S cluster; then, to cytochrome c1; and, finally, to a molecule of oxidized cytochrome c, converting it into its reduced form. The reduced cytochrome c molecule is free to move away from the enzyme to the final complex of the respiratory chain. The second electron passes through two heme groups of cytochrome b to an oxidized ubiquinone in a second Q binding site (Qi). The Q in the second binding site is reduced to a semiquinone radical anion (Q∙− ) by the electron from the first QH2. The now fully oxidized Q leaves the first Q site, free to reenter the Q pool.

A second molecule of QH2 binds to Q-cytochrome c oxidoreductase and reacts in the same way as the first. One of the electrons is transferred to cytochrome c. The second electron passes through the two heme groups of cytochrome b to the partly reduced ubiquinone bound in the second binding site. On the addition of the elec- tron from the second QH2 molecule, this quinone radical anion takes up two pro- tons from the matrix side to form QH2. The removal of these two protons from the matrix contributes to the formation of the proton gradient. This complex set of reactions can be summarized as follows: four protons are released into the intermembrane space, and two protons are removed from the mitochondrial matrix.

In one Q cycle, two QH2 molecules are oxidized to form two Q molecules, and then one Q molecule is reduced to QH2. The problem of how to efficiently funnel electrons from a two-electron carrier (QH2) to a one-electron carrier (cytochrome c) is solved by the Q cycle. The cytochrome b component of the reductase is in essence a recycling device that enables both electrons of QH2 to be used effectively.

Cytochrome c Oxidase Catalyzes the Reduction of Molecular Oxygen to Water

The last of the three proton-pumping assemblies of the respiratory chain is cytochrome c oxidase (Complex IV). Cytochrome c oxidase catalyzes the transfer of electrons from the reduced form of cytochrome c to molecular oxygen, the final acceptor:

The requirement of oxygen for this reaction is what makes “aerobic” organisms aerobic. Obtaining oxygen for this reaction is the primary reason that human beings must breathe. Four electrons are funneled to O 2 to completely reduce it to two molecules of H2O, and, concurrently, protons are pumped from the matrix to the cytoplasmic side of the inner mitochondrial membrane. This reaction is quite thermodynamically favorable. Cytochrome c oxidase, which consists of 13 subunits, contains two heme groups (heme a and heme a3) and three copper ions, arranged as two copper centers (CuA and CuB), with CuA containing two copper ions. Four molecules of reduced cytochrome c generated by Q-cytochrome c oxidoreductase bind consecutively to cytochrome c oxidase and transfer an electron to reduce one molecule of O2 to H2O.

Electrons from two molecules of reduced cytochrome c flow through the oxidation –reduction reactions, one stopping at CuB and the other at heme a3. With both centers in the reduced state, they together can now bind an oxygen molecule.

2. As molecular oxygen binds, it removes an electron from each of the nearby ions in the active center to form a peroxide (O22−) bridge between them. 3. Two more molecules of cytochrome c bind and release electrons that travel to the active center. The addition of an electron as well as H+ to each oxygen atom reduces the two ion–oxygen groups to CuB2+—OH and Fe3+ —OH. 4. Reaction with two more H+ ions allows the release of two molecules of H2O and resets the enzyme to its initial, fully oxidized form:

The four protons in this reaction come exclusively from the matrix. Thus, the consumption of these four protons contributes directly to the proton gradient. Consuming these four protons requires an amount substantially less than the free energy released from the reduction of oxygen to water. What is the fate of this missing energy? Remarkably, cytochrome c oxidase uses this energy to pump four additional protons from the matrix to the cytoplasmic side of the membrane in the course of each reaction cycle for a total of eight protons removed from the matrix. The details of how these protons are transported through the protein is still under study. Thus, the overall process catalyzed by cytochrome c oxidase is

Toxic Derivatives of Molecular Oxygen Such As Superoxide Radical Are Scavenged by Protective Enzymes

Molecular oxygen is an ideal terminal electron acceptor because its high affinity for electrons provides a large thermodynamic driving force. However, the reduction of O2 can result in dangerous side reactions. The transfer of four electrons leads to safe products (two molecules of H2O), but partial reduction generates hazardous compounds. In particular, the transfer of a single electron to O2 forms superoxide ion, whereas the transfer of two electrons yields peroxide:

From 2% to 4% of the oxygen molecules consumed by mitochondria are converted into superoxide ion, predominantly at Complexes I and III. Superoxide, peroxide, and species that can be generated from them such as the hydroxyl radical (O...