Oxidative phosphorylation is a process that involves series of reaction which includes electron transfer from its donor to different acceptor energy is release from this electron transport which will ultimately lead to ATP synthesis. Oxidative phosphorylation is the culmination of energy-yielding metabolism (catabolism) in aerobic organisms. All oxidative steps in the degradation of carbohydrates, fats, and amino acids converge at this final stage of cellular respiration, in which the energy of oxidation drives the synthesis of ATP. Oxidative phosphorylation accounts for most of the ATP synthesized by nonphotosynthetic organisms under most circumstances.
In eukaryotes, oxidative phosphorylation occurs in mitochondria and involves huge protein complexes embedded in the mitochondrial membranes. The pathway to ATP synthesis in mitochondria challenged and fascinated biochemists for much of the twentieth century.
Our current understanding of ATP synthesis in mitochondria is based on the theory, introduced by Peter Mitchell in 1961, that transmembrane differences in proton concentration are the reservoir for the energy extracted from biological oxidation reactions. This chemiosmotic theory has been accepted as one of the great unifying principles of twentieth-century biology. It provides insight into the processes of oxidative phosphorylation and photophosphorylation in plants, and into such apparently disparate energy transductions as active transport across membranes and the motion of bacterial flagella.
The mechanism of oxidative phosphorylation has three defining components.
(1) Electrons flow from electron donors (oxidizable substrates) through a chain of membrane-bound carriers to a final electron acceptor with a large reduction potential (molecular oxygen, O2 ).
(2) The free energy made available by this “downhill” (exergonic) electron flow is coupled to the “uphill” transport of protons across a proton-impermeable membrane, conserving the free energy of fuel oxidation as a transmembrane electrochemical potential.
(3) The transmembrane flow of protons back down their concentration gradient through specific protein channels provides the free energy for synthesis of ATP, catalyzed by a membrane protein complex (ATP synthase) that couples proton flow to phosphorylation of ADP.
Oxidative phosphorylation begins with the entry of electrons into the series of electron carriers called the respiratory chain.
Most of these electrons arise from the action of dehydrogenases that collect electrons from catabolic pathways and funnel them into universal electron acceptors—nicotinamide nucleotides (NAD+ or NADP + ) or flavin nucleotides (FMN or FAD)
The electron carriers of the respiratory chain are organized into membraneembedded supramolecular complexes that can be physically separated. Gentle treatment of the inner mitochondrial membrane with detergents allows the resolution of four unique electron-carrier complexes, each capable of catalyzing electron transfer through a portion of the chain. Complexes I and II catalyze electron transfer to ubiquinone from two different electron donors: NADH (Complex I) and succinate (Complex II). Complex III carries electrons from reduced ubiquinone to cytochrome c, and Complex IV completes the sequence by transferring electrons from cytochrome c to O2 .
We now look in more detail at the structure and function of each complex of the mitochondrial respiratory chain.
- Complex I: NADH to Ubiquinone In mammals, Complex I, also called NADH:ubiquinone oxidoreductase or NADH dehydrogenase, is a large enzyme composed of 45 different polypeptide chains, including an FMNcontaining flavoprotein and at least 8 iron-sulfur centers. Complex I is Lshaped, with one arm embedded in the inner membrane and the other extending into the matrix.
Complex I catalyzes two simultaneous and obligately coupled processes:
(1) the exergonic transfer to ubiquinone of a hydride ion from NADH and a proton from the matrix, expressed by
(2)The endergonic transfer of four protons from the matrix to the intermembrane space. Protons are moved against a transmembrane proton gradient in this process. Complex I is therefore a proton pump driven by the energy of electron transfer
(A) IRON SULPHUR CENTRE
In iron-sulfur proteins, the iron is present not in heme but in association with inorganic sulfur atoms or with the sulfur atoms of Cys residues in the protein, or both. These iron-sulfur (Fe-S) centers range from simple structures with a single Fe atom coordinated to four Cys —SH groups to more complex Fe-S centers with two or four Fe atoms.
Rieske iron-sulfur proteins (named after their discoverer, John S. Rieske) are a variation on this theme, in which one Fe atom is coordinated to two His residues rather than two Cys residues. All iron-sulfur proteins participate in one-electron transfers in which one iron atom of the Fe-S cluster is oxidized or reduced. At least eight Fe-S proteins function in mitochondrial electron transfer. The reduction potential of Fe-S proteins varies from −0.65 V to +0.45 V, depending on the microenvironment of the iron within the protein.
(B) Complex II: Succinate to Ubiquinone Complex II couples the oxidation of succinate at one site with the reduction of ubiquinone at another site about 40 Å away. Although smaller and simpler than Complex I, Complex II contains five prosthetic groups of two types and four different protein subunits. Subunits C and D are integral membrane proteins, each with three transmembrane helices. They contain a heme group, heme b, and a binding site for Q, the final electron acceptor in the reaction catalyzed by Complex II. Subunits A and B extend into the matrix; they contain three 2Fe-2S centers, bound FAD, and a binding site for the substrate, succinate.
Although the overall path of electron transfer is long (from the succinate-binding site to FAD, then through the Fe-S centers to the Q-binding site), none of the individual electron-transfer distances exceeds about 11 Å—a reasonable distance for rapid electron transfer. Electron transfer through Complex II is not accompanied by proton pumping across the inner membrane, although the QH2 produced by succinate oxidation will be used by Complex III to drive proton transfer. Because Complex II functions in the citric acid cycle, factors that affect its activity (such as the availability of oxidized Q) probably serve to coordinate that cycle with mitochondrial electron transfer.
(C) Complex III: Ubiquinone to Cytochrome c Electrons from reduced ubiquinone (ubiquinol, QH2 ) pass through two more large protein complexes in the inner mitochondrial membrane before reaching the ultimate electron acceptor, O2 . Complex III (also called cytochrome bc1 complex or ubiquinone:cytochrome c oxidoreductase) couples the transfer of electrons from ubiquinol to cytochrome c with the vectorial transport of protons from the matrix to the intermembrane space.
The functional unit of Complex III is a dimer. Each monomer consists of three proteins central to the action of the complex: cytochrome b, cytochrome c1 , and the Rieske ironsulfur protein. (Several other proteins associated with Complex III in vertebrates are not conserved across the phyla and presumably play subsidiary roles.)
The two cytochrome b monomers surround a cavern in the middle of the membrane, in which ubiquinone is free to move from the matrix side of the membrane (site QN on one monomer) to the intermembrane space (site QP on the other monomer) as it shuttles electrons and protons across the inner mitochondrial membrane.
To account for the role of Q in energy conservation, Mitchell proposed the “Q cycle”. As electrons move from QH2 through Complex III, QH2 is oxidized with the release of protons on one side of the membrane (at QP ), while at the other site (QN), Q is reduced and protons are taken up. The product of one catalytic site thus becomes the substrate at the second site, and vice versa.
The Q cycle accommodates the switch between the two-electron carrier ubiquinol (the reduced form of ubiquinone) and the one-electron carriers— hemes bL and bH of cytochrome b, and cytochromes c1 and c—and results in the uptake of two protons on the N side and the release of four protons on the P side, per pair of electrons passing through Complex III to cytochrome c.
Two of the protons released on the P side are electrogenic; the other two are electroneutral, balanced by the two charges (electrons) passed to cytochrome c on the P side. Although the path of electrons through this segment of the respiratory chain is complicated, the net effect of the transfer is simple: QH2 is oxidized to Q, two molecules of cytochrome c are reduced, and two protons are moved from the P side to the N side of the inner mitochondrial membrane.
Cytochrome c is a soluble protein of the intermembrane space, which associates reversibly with the P side of the inner membrane. After its single heme accepts an electron from Complex III, cytochrome c moves in the intermembrane space to Complex IV to donate the electron to a binuclear copper center.
(D) Complex IV: Cytochrome c to O2 In the final step of the respiratory chain, Complex IV, also called cytochrome oxidase, carries electrons from cytochrome c to molecular oxygen, reducing it to H2O. Complex IV is a large, dimeric enzyme of the inner mitochondrial membrane,
Subunit II of Complex IV contains two Cu ions complexed with the —SH groups of two Cys residues in a binuclear center that resembles the 2Fe-2S centers of iron-sulfur proteins. Subunit I contains two heme groups, designated a and a3 , and another copper ion (CuB). Heme a3 and CuB form a second binuclear center that accepts electrons from heme a and transfers them to O2 bound to heme a3 .
Electron transfer through Complex IV is from cytochrome c to the CuA center, to heme a, to the heme a3–CuB center, and finally to O2. For every two electrons passing through this complex, the enzyme consumes two “substrate” H+ from the matrix (N side) in converting ½O2 to H2O. It also uses the energy of this redox reaction to pump two protons outward into the intermembrane space (P side) for each pair of electrons that pass through, adding to the electrochemical potential produced by redox-driven proton transport through Complexes I and III.
This two-electron reduction of ½O2 requires the oxidation of QH2 , which in turn requires oxidation of NADH or succinate.
At Complex IV, O2 is reduced at redox centers that carry only one electron at a time. Normally the incompletely reduced oxygen intermediates remain tightly bound to the complex until completely converted to water, but a small fraction of oxygen intermediates escape.
How is a concentration gradient of protons transformed into ATP? Electron transfer releases, and the proton-motive force conserves, more than enough free energy (about 190 kJ) per “mole” of electron pairs to drive the formation of a mole of ATP, which requires about 50 kJ. Mitochondrial oxidative phosphorylation therefore poses no thermodynamic problem. But what is the chemical mechanism that couples proton flux with phosphorylation?
THE CHEMIOSMOTIC MODEL
According to the model , the electrochemical energy inherent in the difference in proton concentration and the separation of charge across the inner mitochondrial membrane—the proton-motive force —drives the synthesis of ATP as protons flow passively back into the matrix through a proton pore in ATP synthase. The overall process is sometimes referred to as “chemiosmotic coupling.” Here, “coupling” refers to the obligate connection between mitochondrial ATP synthesis and electron flow through the respiratory chain; neither of the two processes can proceed without the other.
This large enzyme complex of the inner mitochondrial membrane catalyzes the formation of ATP from ADP and Pi , driven by the flow of protons from the P to the N side of the membrane. ATP synthase, also called Complex V, has two distinct components: F1 , a peripheral membrane protein, and Fo (o denoting oligomycin-sensitive), which is integral to the membrane. F1.
Mitochondrial F1 has nine subunits of five different types, with the composition α3β3γδε. Each of the three β subunits has one catalytic site for ATP synthesis. The crystallographic determination of the F1. The knoblike portion of F1 is a flattened sphere, 8 nm by 10 nm, consisting of alternating α and β subunits. The polypeptides that make up the stalk in the F1 crystal structure are asymmetrically arranged, with one domain of the single γ subunit making up a central shaft that passes through F1.
The Fo complex, with its proton pore, is composed of three subunits, a, b, and c, in the proportion ab2cn.
Paul Boyer proposed a rotational catalysis mechanism in which the three active sites of F1 take turns catalyzing ATP synthesis. A given β subunit starts in the β-ADP conformation, which binds ADP and Pi from the surrounding medium. The subunit now changes conformation, assuming the β-ATP form that tightly binds and stabilizes ATP, bringing about the ready equilibration of ADP + Pi with ATP on the enzyme surface.
Finally, the subunit changes to the β-empty conformation, which has very low affinity for ATP, and the newly synthesized ATP leaves the enzyme surface. Another round of catalysis begins when this subunit again assumes the β-ADP form and binds ADP and Pi. The F1 complex has three nonequivalent adenine nucleotide–binding sites, one for each pair of α and β subunits.
At any given moment, one of these sites is in the β-ATP conformation (which binds ATP tightly), a second is in the β-ADP (loosebinding) conformation, and a third is in the β-empty (very-loose-binding) conformation. In this view from the N side, the proton-motive force causes rotation of the central shaft—the γ subunit, which comes into contact with each αβ subunit pair in succession.
This produces a cooperative conformational change in which the β-ATP site is converted to the β-empty conformation, and ATP dissociates; the β-ADP site is converted to the β-ATP conformation, which promotes condensation of bound ADP + Pi to form ATP; and the β-empty site becomes a β-ADP site, which loosely binds ADP + Pi entering from the solvent.
This model, based on experimental findings, requires that at least two of the three catalytic sites alternate in activity; ATP cannot be released from one site unless and until ADP and Pi are bound at the other.
Note that the direction of rotation reverses when the ATP synthase is acting as an ATPase One strong prediction of this binding-change model is that the γ subunit should rotate in one direction when FoF1 is synthesizing ATP and in the opposite direction when the enzyme is hydrolyzing ATP.
- Chemiosmotic theory provides the intellectual framework for understanding many biological energy transductions, including oxidative phosphorylation and photophosphorylation. The energy of electron flow is conserved by the concomitant pumping of protons across the membrane, producing an electrochemical gradient, the proton-motive force.
- In mitochondria, hydride ions removed from substrates (such as αketoglutarate and malate) by NAD-linked dehydrogenases donate electrons to the respiratory chain, which transfers the electrons to molecular O2 , reducing it to H2O.
- Reducing equivalents from NADH are passed through a series of Fe-S centers to ubiquinone, which transfers the electrons to cytochrome b, the first carrier in Complex III. In this complex, electrons take two separate paths through two b-type cytochromes and cytochrome c1 to an Fe-S center. The Fe-S center passes electrons, one at a time, through cytochrome c and into Complex IV, cytochrome oxidase. This copper-containing enzyme, which also contains cytochromes a and a3 , accumulates electrons, then passes them to O2 , reducing it to H2O.
- Some electrons enter this chain of carriers through alternative paths. Succinate is oxidized by succinate dehydrogenase (Complex II), which contains a flavoprotein that passes electrons through several Fe-S centers to ubiquinone. Electrons derived from the oxidation of fatty acids pass to ubiquinone via the electron-transferring flavoprotein. The oxidation of glycerol phosphate and of dihydroorotate also sends electrons into the respiratory chain at the level of QH2 .
- Potentially harmful reactive oxygen species produced in mitochondria are inactivated by a set of protective enzymes, including superoxide dismutase and glutathione peroxidase. Low levels of ROS serve as signals coordinating mitochondrial oxidative phosphorylation with other metabolic pathways.
- Plants, fungi, and unicellular eukaryotes have, in addition to the typical path for electron transfer coupled to ATP synthesis, an alternative, uncoupled pathway that recycles excess NADH to NAD+ .
- The flow of electrons through Complexes I, III, and IV results in pumping of protons across the inner mitochondrial membrane, making the matrix alkaline relative to the intermembrane space. This proton gradient provides the energy, in the form of the proton-motive force, for ATP synthesis from ADP and Pi by ATP synthase (FoF1 complex) in the inner membrane.
- ATP synthase carries out “rotational catalysis,” in which the flow of protons through Fo causes each of three nucleotide-binding sites in F1 to cycle from (ADP + Pi )–bound to ATP-bound to empty conformations.
- ATP formation on the enzyme requires little energy; the role of the protonmotive force is to push ATP from its binding site on the synthase.
- The ratio of ATP synthesized per ½O2 reduced to H2O (the P/O ratio) is about 2.5 when electrons enter the respiratory chain at Complex I, and 1.5 when electrons enter at ubiquinone. This ratio varies among species, depending on the number of c subunits in the Fo complex.
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:- Article Written By Zahra Madraswala