Oxidative phosphorylation[N 1] orelectron transport-linked phosphorylation orterminal oxidation, is themetabolic pathway in whichcells useenzymes tooxidizenutrients, thereby releasing chemical energy in order to produceadenosine triphosphate (ATP). Ineukaryotes, this takes place insidemitochondria. Almost allaerobic organisms carry out oxidative phosphorylation. This pathway is so pervasive because it releases more energy thanfermentation.
Inaerobic respiration, the energy stored in the chemical bonds ofglucose is released by the cell inglycolysis and subsequently thecitric acid cycle, producing carbon dioxide and the energeticelectron donorsNADH andFADH. Oxidative phosphorylation uses these molecules and O2 toproduce ATP, which is used throughout the cell whenever energy is needed. During oxidative phosphorylation, electrons are transferred from the electron donors to a series ofelectron acceptors in a series ofredox reactions ending in oxygen, whose reaction releases half of the total energy.[1]
Ineukaryotes, these redox reactions are catalyzed by a series ofprotein complexes within theinner mitochondrial membrane; whereas, inprokaryotes, these proteins are located in the cell'splasma membrane. These linked sets of proteins are called theelectron transport chain. In mitochondria, five main protein complexes are involved, whereas prokaryotes have various other enzymes, using a variety of electron donors and acceptors.
The energy transferred by electrons flowing through this electron transport chain is used to transportprotons across the inner membrane. This generatespotential energy in the form of apH gradient and the resultingelectrical potential across this membrane. This store of energy is tapped when protons flow back across the membrane throughATP synthase in a process calledchemiosmosis. The ATP synthase uses the energy to transformadenosine diphosphate (ADP) into adenosine triphosphate, in aphosphorylation reaction. The reaction is driven by the proton flow, which forces therotation of a part of the enzyme. The ATP synthase is a rotary mechanical motor.
Although oxidative phosphorylation is a vital part of metabolism, it producesreactive oxygen species such assuperoxide andhydrogen peroxide, which lead to propagation offree radicals, damaging cells and contributing todisease and, possibly,aging andsenescence. The enzymes carrying out this metabolic pathway are also the target of many drugs and poisons thatinhibit their activities.
Oxidative phosphorylation works by usingenergy-releasing chemical reactions to drive energy-requiring reactions. The two sets of reactions are said to becoupled. This means one cannot occur without the other. The chain of redox reactions driving the flow of electrons through the electron transport chain, from electron donors such asNADH toelectron acceptors such asoxygen and hydrogen (protons), is anexergonic process – it releases energy, whereas the synthesis of ATP is anendergonic process, which requires an input of energy. Both the electron transport chain and the ATP synthase are embedded in a membrane, and energy is transferred from the electron transport chain to the ATP synthase by movements of protons across this membrane, in a process calledchemiosmosis.[2] A current of protons is driven from the negative N-side of the membrane to the positive P-side through the proton-pumping enzymes of the electron transport chain. The movement of protons creates anelectrochemical gradient across the membrane, is called theproton-motive force. It has two components: a difference in proton concentration (a H+ gradient, ΔpH) and a difference inelectric potential, with the N-side having a negative charge.[3]
ATP synthase releases this stored energy by completing the circuit and allowing protons to flow down the electrochemical gradient, back to the N-side of the membrane.[4] The electrochemical gradient drives the rotation of part of the enzyme's structure and couples this motion to the synthesis of ATP.
The two components of the proton-motive force arethermodynamically equivalent: In mitochondria, the largest part of energy is provided by the potential; inalkaliphile bacteria the electrical energy even has to compensate for a counteracting inverse pH difference. Inversely,chloroplasts operate mainly on ΔpH. However, they also require a small membrane potential for the kinetics of ATP synthesis. In the case of thefusobacteriumPropionigenium modestum it drives the counter-rotation of subunits a and c of the FO motor of ATP synthase.[3]
The amount of energy released by oxidative phosphorylation is high, compared with the amount produced byfermentation.Glycolysis produces only 2 ATP molecules, but somewhere between 30 and 36 ATPs are produced by the oxidative phosphorylation of the 10 NADH and 2 succinate molecules made by converting one molecule ofglucose to carbon dioxide and water,[5] while each cycle ofbeta oxidation of afatty acid yields about 14 ATPs. These ATP yields are theoretical maximum values; in practice, some protons leak across the membrane, lowering the yield of ATP.[6]
The electron transport chain carries both protons and electrons, passing electrons from donors to acceptors, and transporting protons across a membrane. These processes use both soluble and protein-bound transfer molecules. In the mitochondria, electrons are transferred within the intermembrane space by the water-soluble electron transfer proteincytochrome c.[7] This carries only electrons, and these are transferred by the reduction and oxidation of aniron atom that the protein holds within aheme group in its structure. Cytochrome c is also found in some bacteria, where it is located within theperiplasmic space.[8]
Within the inner mitochondrial membrane, thelipid-soluble electron carriercoenzyme Q10 (Q) carries both electrons and protons by aredox cycle.[9] This smallbenzoquinone molecule is veryhydrophobic, so it diffuses freely within the membrane. When Q accepts two electrons and two protons, it becomes reduced to theubiquinol form (QH2); when QH2 releases two electrons and two protons, it becomes oxidized back to theubiquinone (Q) form. As a result, if two enzymes are arranged so that Q is reduced on one side of the membrane and QH2 oxidized on the other, ubiquinone will couple these reactions and shuttle protons across the membrane.[10] Some bacterial electron transport chains use different quinones, such asmenaquinone, in addition to ubiquinone.[11]
Within proteins, electrons are transferred betweenflavin cofactors,[4][12]iron–sulfur clusters and cytochromes. There are several types of iron–sulfur cluster. The simplest kind found in the electron transfer chain consists of two iron atoms joined by two atoms of inorganicsulfur; these are called [2Fe–2S] clusters. The second kind, called [4Fe–4S], contains a cube of four iron atoms and four sulfur atoms. Each iron atom in these clusters is coordinated by an additionalamino acid, usually by the sulfur atom ofcysteine. Metal ion cofactors undergo redox reactions without binding or releasing protons, so in the electron transport chain they serve solely to transport electrons through proteins. Electrons move quite long distances through proteins by hopping along chains of these cofactors.[13] This occurs byquantum tunnelling, which is rapid over distances of less than 1.4×10−9 m.[14]
Manycatabolic biochemical processes, such asglycolysis, thecitric acid cycle, andbeta oxidation, produce the reducedcoenzymeNADH. This coenzyme contains electrons that have a hightransfer potential; in other words, they will release a large amount of energy upon oxidation. However, the cell does not release this energy all at once, as this would be an uncontrollable reaction. Instead, the electrons are removed from NADH and passed to oxygen through a series of enzymes that each release a small amount of the energy. This set of enzymes, consisting of complexes I through IV, is called the electron transport chain and is found in theinner membrane of the mitochondrion.Succinate is also oxidized by the electron transport chain, but feeds into the pathway at a different point.
Ineukaryotes, the enzymes in this electron transport system use the energy released from O2 by NADH to pumpprotons across the inner membrane of the mitochondrion. This causes protons to build up in theintermembrane space, and generates anelectrochemical gradient across the membrane. The energy stored in this potential is then used by ATP synthase to produce ATP. Oxidative phosphorylation in the eukaryotic mitochondrion is the best-understood example of this process. The mitochondrion is present in almost all eukaryotes, with the exception of anaerobic protozoa such asTrichomonas vaginalis that instead reduce protons to hydrogen in a remnant mitochondrion called ahydrogenosome.[15]
| Respiratory enzyme | Redox pair | Midpoint potential (Volts) |
|---|---|---|
| NADH dehydrogenase | NAD+ /NADH | −0.32[16] |
| Succinate dehydrogenase | FMN orFAD / FMNH2 or FADH2 | −0.20[16] |
| Cytochrome bc1 complex | Coenzyme Q10ox / Coenzyme Q10red | +0.06[16] |
| Cytochrome bc1 complex | Cytochrome box / Cytochrome bred | +0.12[16] |
| Complex IV | Cytochrome cox / Cytochrome cred | +0.22[16] |
| Complex IV | Cytochrome aox / Cytochrome ared | +0.29[16] |
| Complex IV | O2 / HO− | +0.82[16] |
| Conditions: pH = 7[16] | ||
NADH-coenzyme Q oxidoreductase, also known asNADH dehydrogenase orcomplex I, is the first protein in the electron transport chain.[17] Complex I is a giantenzyme with the mammalian complex I having 46 subunits and a molecular mass of about1000 kDa.[18] The structure is known in detail only from a bacterium;[19][20] in most organisms the complex resembles a boot with a large "ball" poking out from the membrane into the mitochondrion.[21][22] The genes that encode the individual proteins are contained in both thecell nucleus and themitochondrial genome, as is the case for many enzymes present in the mitochondrion.
The reaction that is catalyzed by this enzyme is the two electron oxidation ofNADH bycoenzyme Q10 orubiquinone (represented as Q in the equation below), a lipid-solublequinone that is found in the mitochondrion membrane:
| 1 |
The start of the reaction, and indeed of the entire electron chain, is the binding of a NADH molecule to complex I and the donation of two electrons. The electrons enter complex I via aprosthetic group attached to the complex,flavin mononucleotide (FMN). The addition of electrons to FMN converts it to its reduced form, FMNH2. The electrons are then transferred through a series of iron–sulfur clusters: the second kind of prosthetic group present in the complex.[19] There are both [2Fe–2S] and [4Fe–4S] iron–sulfur clusters in complex I.
As the electrons pass through this complex, four protons are pumped from the matrix into the intermembrane space. Exactly how this occurs is unclear, but it seems to involveconformational changes in complex I that cause the protein to bind protons on the N-side of the membrane and release them on the P-side of the membrane.[23] Finally, the electrons are transferred from the chain of iron–sulfur clusters to a ubiquinone molecule in the membrane.[17] Reduction of ubiquinone also contributes to the generation of a proton gradient, as two protons are taken up from the matrix as it is reduced toubiquinol (QH2).
Succinate-Q oxidoreductase, also known ascomplex II orsuccinate dehydrogenase, is a second entry point to the electron transport chain.[24] It is unusual because it is the only enzyme that is part of both the citric acid cycle and the electron transport chain. Complex II consists of four protein subunits and contains a boundflavin adenine dinucleotide (FAD) cofactor, iron–sulfur clusters, and aheme group that does not participate in electron transfer to coenzyme Q, but is believed to be important in decreasing production of reactive oxygen species.[25][26] It oxidizessuccinate tofumarate and reduces ubiquinone. As this reaction releases less energy than the oxidation of NADH, complex II does not transport protons across the membrane and does not contribute to the proton gradient.
| 2 |
In some eukaryotes, such as theparasitic wormAscaris suum, an enzyme similar to complex II, fumarate reductase (menaquinol:fumarateoxidoreductase, or QFR), operates in reverse to oxidize ubiquinol and reduce fumarate. This allows the worm to survive in the anaerobic environment of thelarge intestine, carrying out anaerobic oxidative phosphorylation with fumarate as the electron acceptor.[27] Another unconventional function of complex II is seen in themalaria parasitePlasmodium falciparum. Here, the reversed action of complex II as an oxidase is important in regenerating ubiquinol, which the parasite uses in an unusual form ofpyrimidine biosynthesis.[28]
Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-Q oxidoreductase), also known aselectron transferring-flavoprotein dehydrogenase, is a third entry point to the electron transport chain. It is an enzyme that accepts electrons fromelectron-transferring flavoprotein in the mitochondrial matrix, and uses these electrons to reduce ubiquinone.[29] This enzyme contains aflavin and a [4Fe–4S] cluster, but, unlike the other respiratory complexes, it attaches to the surface of the membrane and does not cross the lipid bilayer.[30]
| 3 |
In mammals, this metabolic pathway is important inbeta oxidation offatty acids and catabolism ofamino acids andcholine, as it accepts electrons from multipleacetyl-CoA dehydrogenases.[31][32] In plants, ETF-Q oxidoreductase is also important in the metabolic responses that allow survival in extended periods of darkness.[33]
Q-cytochrome c oxidoreductase is also known ascytochrome c reductase,cytochrome bc1 complex, or simplycomplex III.[34][35] In mammals, this enzyme is adimer, with each subunit complex containing 11 protein subunits, an [2Fe-2S] iron–sulfur cluster and threecytochromes: onecytochrome c1 and two bcytochromes.[36] A cytochrome is a kind of electron-transferring protein that contains at least oneheme group. The iron atoms inside complex III's heme groups alternate between a reduced ferrous (+2) and oxidized ferric (+3) state as the electrons are transferred through the protein.
The reaction catalyzed by complex III is the oxidation of one molecule ofubiquinol and the reduction of two molecules ofcytochrome c, a heme protein loosely associated with the mitochondrion. Unlike coenzyme Q, which carries two electrons, cytochrome c carries only one electron.
| 4 |
As only one of the electrons can be transferred from the QH2 donor to a cytochrome c acceptor at a time, the reaction mechanism of complex III is more elaborate than those of the other respiratory complexes, and occurs in two steps called theQ cycle.[37] In the first step, the enzyme binds three substrates, first, QH2, which is then oxidized, with one electron being passed to the second substrate, cytochrome c. The two protons released from QH2 pass into the intermembrane space. The third substrate is Q, which accepts the second electron from the QH2 and is reduced to Q.−, which is theubisemiquinonefree radical. The first two substrates are released, but this ubisemiquinone intermediate remains bound. In the second step, a second molecule of QH2 is bound and again passes its first electron to a cytochrome c acceptor. The second electron is passed to the bound ubisemiquinone, reducing it to QH2 as it gains two protons from the mitochondrial matrix. This QH2 is then released from the enzyme.[38]
As coenzyme Q is reduced to ubiquinol on the inner side of the membrane and oxidized to ubiquinone on the other, a net transfer of protons across the membrane occurs, adding to the proton gradient.[4] The rather complex two-step mechanism by which this occurs is important, as it increases the efficiency of proton transfer. If, instead of the Q cycle, one molecule of QH2 were used to directly reduce two molecules of cytochrome c, the efficiency would be halved, with only one proton transferred per cytochrome c reduced.[4]
Cytochrome c oxidase, also known ascomplex IV, is the final protein complex in the electron transport chain.[39] The mammalian enzyme has an extremely complicated structure and contains 13 subunits, two heme groups, as well as multiple metal ion cofactors – in all, three atoms ofcopper, one ofmagnesium and one ofzinc.[40]
This enzyme mediates the final reaction in the electron transport chain and transfers electrons to oxygen and hydrogen (protons), while pumping protons across the membrane.[41] The finalelectron acceptor oxygen is reduced to water in this step. Both the direct pumping of protons and the consumption of matrix protons in the reduction of oxygen contribute to the proton gradient. The reaction catalyzed is the oxidation of cytochrome c and the reduction of oxygen:
| 5 |
Many eukaryotic organisms have electron transport chains that differ from the much-studied mammalian enzymes described above. For example,plants have alternative NADH oxidases, which oxidize NADH in the cytosol rather than in the mitochondrial matrix, and pass these electrons to the ubiquinone pool.[42] These enzymes do not transport protons, and, therefore, reduce ubiquinone without altering the electrochemical gradient across the inner membrane.[43]
Another example of a divergent electron transport chain is thealternative oxidase, which is found inplants, as well as somefungi,protists, and possibly some animals.[44][45] This enzyme transfers electrons directly from ubiquinol to oxygen.[46]
The electron transport pathways produced by these alternative NADH and ubiquinone oxidases have lowerATP yields than the full pathway. The advantages produced by a shortened pathway are not entirely clear. However, the alternative oxidase is produced in response to stresses such as cold,reactive oxygen species, and infection by pathogens, as well as other factors that inhibit the full electron transport chain.[47][48] Alternative pathways might, therefore, enhance an organism's resistance to injury, by reducingoxidative stress.[49]
The original model for how the respiratory chain complexes are organized was that they diffuse freely and independently in the mitochondrial membrane.[50] However, recent data suggest that the complexes might form higher-order structures called supercomplexes or "respirasomes".[51] In this model, the various complexes exist as organized sets of interacting enzymes.[52] These associations might allow channeling of substrates between the various enzyme complexes, increasing the rate and efficiency of electron transfer.[53] Within such mammalian supercomplexes, some components would be present in higher amounts than others, with some data suggesting a ratio between complexes I/II/III/IV and the ATP synthase of approximately 1:1:3:7:4.[54] However, the debate over this supercomplex hypothesis is not completely resolved, as some data do not appear to fit with this model.[18][55]
In contrast to the general similarity in structure and function of the electron transport chains in eukaryotes,bacteria andarchaea possess a large variety of electron-transfer enzymes. These use an equally wide set of chemicals as substrates.[56] In common with eukaryotes, prokaryotic electron transport uses the energy released from the oxidation of a substrate to pump ions across a membrane and generate an electrochemical gradient. For bacteria, oxidative phosphorylation is understood in most detail inEscherichia coli, while archaeal systems are, at present, poorly understood.[57]
The main difference between eukaryotic and prokaryotic oxidative phosphorylation is that bacteria and archaea use many different substances to donate or accept electrons. This allows prokaryotes to grow under a wide variety of environmental conditions.[58] InE. coli, for example, oxidative phosphorylation can be driven by a large number of pairs of reducing agents and oxidizing agents, which are listed below. Themidpoint potential of a chemical measures how much energy is released when it is oxidized or reduced, with reducing agents having negative potentials and oxidizing agents positive potentials.
| Respiratory enzyme | Redox pair | Midpoint potential (Volts) |
|---|---|---|
| Ubiquinol oxidase | Oxygen /Water | +0.82 |
| Nitrate reductase | Nitrate /Nitrite | +0.42 |
| Nitrite reductase | Nitrite /Ammonia | +0.36 |
| Dimethyl sulfoxide reductase | DMSO /DMS | +0.16 |
| TrimethylamineN-oxide reductase | TMAO /TMA | +0.13 |
| Fumarate reductase | Fumarate /Succinate | +0.03 |
| Succinate dehydrogenase | Fumarate /Succinate | +0.03 |
| Glucose dehydrogenase | Gluconate /Glucose | −0.14 |
| Glycerol-3-phosphate dehydrogenase | DHAP /Gly-3-P | −0.19 |
| Lactate dehydrogenase | Pyruvate /Lactate | −0.19 |
| NADH dehydrogenase | NAD+ /NADH | −0.32 |
| Hydrogenase | Proton /Hydrogen | −0.42 |
| Formate dehydrogenase | Bicarbonate /Formate | −0.43 |
| D-amino acid dehydrogenase | 2-oxoacid +ammonia /D-amino acid | ? |
| Pyruvate oxidase | Acetate +Carbon dioxide /Pyruvate | ? |
As shown above,E. coli can grow with reducing agents such as formate, hydrogen, or lactate as electron donors, and nitrate, DMSO, or oxygen as acceptors.[58] The larger the difference in midpoint potential between an oxidizing and reducing agent, the more energy is released when they react. Out of these compounds, the succinate/fumarate pair is unusual, as its midpoint potential is close to zero. Succinate can therefore be oxidized to fumarate if a strong oxidizing agent such as oxygen is available, or fumarate can be reduced to succinate using a strong reducing agent such as formate. These alternative reactions are catalyzed bysuccinate dehydrogenase andfumarate reductase, respectively.[60]
Some prokaryotes use redox pairs that have only a small difference in midpoint potential. For example,nitrifying bacteria such asNitrobacter oxidize nitrite to nitrate, donating the electrons to oxygen. The small amount of energy released in this reaction is enough to pump protons and generate ATP, but not enough to produce NADH or NADPH directly for use inanabolism.[61] This problem is solved by using anitrite oxidoreductase to produce enough proton-motive force to run part of the electron transport chain in reverse, causing complex I to generate NADH.[62][63]
Prokaryotes control their use of these electron donors and acceptors by varying which enzymes are produced in response to environmental conditions.[64] This flexibility is possible because different oxidases and reductases use the same ubiquinone pool. This allows many combinations of enzymes to function together, linked by the common ubiquinol intermediate.[59] These respiratory chains therefore have amodular design, with easily interchangeable sets of enzyme systems.
In addition to this metabolic diversity, prokaryotes also possess a range ofisozymes – different enzymes that catalyze the same reaction. For example, inE. coli, there are two different types of ubiquinol oxidase using oxygen as an electron acceptor. Under highly aerobic conditions, the cell uses an oxidase with a low affinity for oxygen that can transport two protons per electron. However, if levels of oxygen fall, they switch to an oxidase that transfers only one proton per electron, but has a high affinity for oxygen.[65]
ATP synthase, also calledcomplex V, is the final enzyme in the oxidative phosphorylation pathway. This enzyme is found in all forms of life and functions in the same way in both prokaryotes and eukaryotes.[66] The enzyme uses the energy stored in a proton gradient across a membrane to drive the synthesis of ATP from ADP andphosphate (Pi). Estimates of the number of protons required to synthesize one ATP have ranged from three to four,[67][68] with some suggesting cells can vary this ratio, to suit different conditions.[69]
| 6 |
Thisphosphorylation reaction is anequilibrium, which can be shifted by altering the proton-motive force. In the absence of a proton-motive force, the ATP synthase reaction will run from right to left, hydrolyzing ATP and pumping protons out of the matrix across the membrane. However, when the proton-motive force is high, the reaction is forced to run in the opposite direction; it proceeds from left to right, allowing protons to flow down their concentration gradient and turning ADP into ATP.[66] Indeed, in the closely relatedvacuolar type H+-ATPases, the hydrolysis reaction is used to acidify cellular compartments, by pumping protons and hydrolysing ATP.[70]
ATP synthase is a massive protein complex with a mushroom-like shape. The mammalian enzyme complex contains 16 subunits and has a mass of approximately 600kilodaltons.[71] The portion embedded within the membrane is called FO and contains a ring of c subunits and the proton channel. The stalk and the ball-shaped headpiece is called F1 and is the site of ATP synthesis. The ball-shaped complex at the end of the F1 portion contains six proteins of two different kinds (three α subunits and three β subunits), whereas the "stalk" consists of one protein: the γ subunit, with the tip of the stalk extending into the ball of α and β subunits.[72] Both the α and β subunits bind nucleotides, but only the β subunits catalyze the ATP synthesis reaction. Reaching along the side of the F1 portion and back into the membrane is a long rod-like subunit that anchors the α and β subunits into the base of the enzyme.
As protons cross the membrane through the channel in the base of ATP synthase, the FO proton-driven motor rotates.[73] Rotation might be caused by changes in theionization of amino acids in the ring of c subunits causingelectrostatic interactions that propel the ring of c subunits past the proton channel.[74] This rotating ring in turn drives the rotation of the centralaxle (the γ subunit stalk) within the α and β subunits. The α and β subunits are prevented from rotating themselves by the side-arm, which acts as astator. This movement of the tip of the γ subunit within the ball of α and β subunits provides the energy for the active sites in the β subunits to undergo a cycle of movements that produces and then releases ATP.[75]
This ATP synthesis reaction is called thebinding change mechanism and involves the active site of a β subunit cycling between three states.[76] In the "open" state, ADP and phosphate enter the active site (shown in brown in the diagram). The protein then closes up around the molecules and binds them loosely – the "loose" state (shown in red). The enzyme then changes shape again and forces these molecules together, with the active site in the resulting "tight" state (shown in pink) binding the newly produced ATP molecule with very highaffinity. Finally, the active site cycles back to the open state, releasing ATP and binding more ADP and phosphate, ready for the next cycle.
In some bacteria and archaea, ATP synthesis is driven by the movement of sodium ions through the cell membrane, rather than the movement of protons.[77][78] Archaea such asMethanococcus also contain the A1Ao synthase, a form of the enzyme that contains additional proteins with little similarity in sequence to other bacterial and eukaryotic ATP synthase subunits. It is possible that, in some species, the A1Ao form of the enzyme is a specialized sodium-driven ATP synthase,[79] but this might not be true in all cases.[78]
The transport of electrons from redox pair NAD+/ NADH to the final redox pair 1/2 O2/ H2O can be summarized as
1/2 O2 + NADH + H+ → H2O + NAD+
The potential difference between these two redox pairs is 1.14 volt, which is equivalent to -52 kcal/mol or -2600 kJ per 6 mol of O2.
When one NADH is oxidized through the electron transfer chain, three ATPs are produced, which is equivalent to 7.3 kcal/mol x 3 = 21.9 kcal/mol.
The conservation of the energy can be calculated by the following formula
Efficiency = (21.9 x 100%) / 52 = 42%
So we can conclude that when NADH is oxidized, about 42% of energy is conserved in the form of three ATPs and the remaining (58%) energy is lost as heat (unless the chemical energy of ATP under physiological conditions was underestimated).
Molecular oxygen is a good terminalelectron acceptor because it is a strong oxidizing agent. The reduction of oxygen does involve potentially harmful intermediates.[80] Although the transfer of four electrons and four protons reduces oxygen to water, which is harmless, transfer of one or two electrons producessuperoxide orperoxide anions, which are dangerously reactive.
| 7 |
Thesereactive oxygen species and their reaction products, such as thehydroxyl radical, are very harmful to cells, as they oxidize proteins and causemutations inDNA. This cellular damage may contribute todisease and is proposed as one cause ofaging.[81][82]
The cytochrome c oxidase complex is highly efficient at reducing oxygen to water, and it releases very few partly reduced intermediates; however small amounts of superoxide anion and peroxide are produced by the electron transport chain.[83] Particularly important is the reduction ofcoenzyme Q in complex III, as a highly reactive ubisemiquinone free radical is formed as an intermediate in the Q cycle. This unstable species can lead to electron "leakage" when electrons transfer directly to oxygen, forming superoxide.[84] As the production of reactive oxygen species by these proton-pumping complexes is greatest at high membrane potentials, it has been proposed that mitochondria regulate their activity to maintain the membrane potential within a narrow range that balances ATP production against oxidant generation.[85] For instance, oxidants can activateuncoupling proteins that reduce membrane potential.[86]
To counteract these reactive oxygen species, cells contain numerousantioxidant systems, including antioxidantvitamins such asvitamin C andvitamin E, and antioxidant enzymes such assuperoxide dismutase,catalase, andperoxidases,[80] which detoxify the reactive species, limiting damage to the cell.
Asoxygen is fundamental for oxidative phosphorylation, a shortage in O2 level can alter ATP production rates. The proton motive force and ATP production can be maintained by intracellular acidosis.[87] Cytosolic protons that have accumulated with ATP hydrolysis andlactic acidosis can freely diffuse across the mitochondrial outer-membrane and acidify the inter-membrane space, hence directly contributing to the proton motive force and ATP production.
When exposed tohypoxia/anoxia (no oxygen), most animals will see damage done to their mitochondria.[88] From some species, these conditions can happen due to environmental variables, such as low tides,[89] low temperatures,[90] or general living conditions, like living in a hypoxic underground burrow.[91] In humans, these conditions are commonly met in medical emergencies such asstrokes,ischemia, andasphyxia.
Despite this, or perhaps due to it, some species have developed their own defense mechanisms against anoxia/hypoxia, as well as duringreperfusion/reoxygenation. These mechanisms are diverse and differ betweenendotherms andectotherms and can differ even at the species level.
Most mammals and birds are intolerant to low/no oxygen conditions. For the heart, in the absence of oxygen, the first fourcomplexes of the electron transport chain decrease in activity.[88] This will lead to protons leaking through theinner mitochondrial membrane without complexesI,III, andIV pushing protons back through to maintain the proton gradient. There is also electron leak (an event where electrons leak out of the electron transport chain), which happens becauseNADH dehydrogenase within Complex I becomes damaged, which allows for the production of ROS (reactive oxygen species) during ischemia.[92] This will lead to the reversing ofComplex V, which forces protons from thematrix back into theinner membrane space, against theirconcentration gradient. Forcing protons against their concentration gradient requires energy, so Complex V uses up ATP as an energy source.[93]
When oxygen re-enters the system, animals are faced with a different set of problems. Since ATP was used up during the anoxic period, it leads to a lack ofADP within the system.[94] This is due to ADP's natural degradation into AMP, resulting in ADP being drained from the system. With no ADP in the system, Complex V is unable to start, meaning the protons will not flow through it to enter the matrix.[94] Due to Complex V's reversal during anoxia, the proton gradient has become hyperpolarized (where the proton gradient is highly positively charged). Another factor in this problem is thatsuccinate built up during anoxia, so when oxygen is reintroduced, succinate donates electrons toComplex II.[95][96] The hyperpolarized gradient and succinate buildup leads toreverse electron transport, causingoxidative stress,[97] which can lead to cellular damage and diseases.[98]
The naked mole rat (Heterocephalus glaber) is a hypoxia-tolerant species that sleeps in deep burrows and in large colonies. The depth of these burrows reduces access to oxygen, and sleeping in large groups will deplete the area of oxygen quicker than usual, leading to hypoxia.[91] The naked mole rat has the unique ability to survive low oxygen conditions for no less than several hours, and zero oxygen conditions for 18 minutes.[99] One of the ways of combatting hypoxia in the brain is decreasing the reliance on oxygen for ATP production, achieved by decreased respiration rates and proton leak.[91]
Hypoxia/anoxia tolerant species handle ROS production during reoxygenation better than the intolerant. In the cortex of the naked mole rats, they show better homeostasis of ROS production than intolerant species and seem to lack the burst of ROS that typically comes with reoxygenation.[99]
Research on intolerant ectotherms is more limited than on tolerant ectotherms and intolerant endotherms, but it is shown that anoxia/hypoxia intolerance is different in terms for how long the intolerant survive as opposed to the tolerant between endotherms and ectotherms. While intolerant endotherms only last minutes, intolerant ectotherms can last hours, such as subtidal scallops (Argopecten irradians).[100] This difference in intolerance could be due to a couple of different factors. One advantage is that the ectothermic inner mitochondrial membrane is less leaky, so less protons will leak through the inner membrane due to differences in thephospholipid bilayer composition.[93] Another advantage ectotherms tend to have in this category is an ability for their mitochondria to properly function in a wide range of temperatures, such as the western fence lizard (Sceloporus occidentalis). While western fence lizards are not considered a hypoxia-tolerant animal, they still showed less temperature sensitivity in their mitochondria than mice mitochondria.[101]
While it is unclear how reoxygenation affects intolerant ectotherms at the mitochondrial level, there is some research showing how some of them respond. In the hypoxia-sensitive shovelnose ray (Aptychotrema rostrata), it is shown that ROS production is lower upon reoxygenation compared to rays only exposed to normoxia (normal oxygen levels).[89] This differs from the hypoxia-sensitive endotherm, which would see an increase in ROS production. However, the ray's levels were still higher than the more hypoxia-tolerant Epaulette shark (Hemiscyllum ocellatum), which potentially sees hypoxia due to the bouts of low tides that can be seen in reef platforms.[89] Subtidal scallops will see both a decrease in maximal respiration and a depolarization of the membrane during reoxygenation.[100]
Hypoxia/Anoxia tolerant ectotherms have shown unique strategies for surviving anoxia. Pond turtles, such as the painted turtle (Chrysemys picta bellii), will experience anoxia during winter while they overwinter at the bottom of frozen ponds.[90] In their cardiac mitochondria, the reversing of Complex V,[102] the usage of ATP, and the build-up of succinate are all prevented during anoxia.[94] Crucian carps (Carassius carassius) also overwinter in frozen ponds and show no loss membrane potential in their cardiac mitochondria during anoxia, but this relies on complexes I and III to be active.[103]
Pond turtles are able to completely avoid ROS production upon reoxygenation.[104] However, crucian carp cannot and are unable to prevent the death of brain cells upon reoxygenation.[105]
There are several well-knowndrugs andtoxins that inhibit oxidative phosphorylation. Although any one of these toxins inhibits only one enzyme in the electron transport chain, inhibition of any step in this process will halt the rest of the process. For example, ifoligomycin inhibits ATP synthase, protons cannot pass back into the mitochondrion.[106] As a result, the proton pumps are unable to operate, as the gradient becomes too strong for them to overcome. NADH is then no longer oxidized and the citric acid cycle ceases to operate because the concentration of NAD+ falls below the concentration that these enzymes can use.
Many site-specific inhibitors of the electron transport chain have contributed to the present knowledge of mitochondrial respiration. Synthesis of ATP is also dependent on the electron transport chain, so all site-specific inhibitors also inhibit ATP formation. The fish poisonrotenone, the barbiturate drugamytal, and the antibioticpiericidin A inhibit NADH and coenzyme Q.[107]
Carbon monoxide,cyanide,hydrogen sulfide andazide effectively inhibit cytochrome oxidase. Carbon monoxide reacts with the reduced form of the cytochrome while cyanide and azide react with the oxidised form. An antibiotic,antimycin A, andBritish anti-Lewisite, an antidote used against chemical weapons, are the two important inhibitors of the site between cytochrome B and C1.[107]
| Compounds | Use | Site of action | Effect on oxidative phosphorylation |
|---|---|---|---|
| Cyanide Carbon monoxide Azide Hydrogen sulfide | Poisons | Complex IV | Inhibit the electron transport chain by binding more strongly than oxygen to theFe–Cu center in cytochrome c oxidase, preventing the reduction of oxygen.[108] |
| Oligomycin | Antibiotic | Complex V | Inhibits ATP synthase by blocking the flow of protons through the Fo subunit.[106] |
| CCCP 2,4-Dinitrophenol | Poisons, weight-loss[N 2] | Inner membrane | Ionophores that disrupt the proton gradient by carrying protons across a membrane. This ionophoreuncouples proton pumping from ATP synthesis because it carries protons across the inner mitochondrial membrane.[109] |
| Rotenone | Pesticide | Complex I | Prevents the transfer of electrons from complex I to ubiquinone by blocking the ubiquinone-binding site.[110] |
| Malonate andoxaloacetate | Poisons | Complex II | Competitive inhibitors of succinate dehydrogenase (complex II).[111] |
| Antimycin A | Piscicide | Complex III | Binds to the Qi site ofcytochrome c reductase, thereby inhibiting theoxidation ofubiquinol. |
Not all inhibitors of oxidative phosphorylation are toxins. Inbrown adipose tissue, regulated proton channels calleduncoupling proteins can uncouple respiration from ATP synthesis.[112] This rapid respiration produces heat, and is particularly important as a way of maintainingbody temperature forhibernating animals, although these proteins may also have a more general function in cells' responses to stress.[113]
The field of oxidative phosphorylation began with the report in 1906 byArthur Harden of a vital role for phosphate in cellularfermentation, but initially onlysugar phosphates were known to be involved.[114] However, in the early 1940s, the link between the oxidation of sugars and the generation of ATP was firmly established byHerman Kalckar,[115] confirming the central role of ATP in energy transfer that had been proposed byFritz Albert Lipmann in 1941.[116] Later, in 1949, Morris Friedkin andAlbert L. Lehninger proved that the coenzyme NADH linked metabolic pathways such as the citric acid cycle and the synthesis of ATP.[117] The termoxidative phosphorylation was coined byVolodymyr Belitser [uk] in 1939.[118][119]
For another twenty years, the mechanism by which ATP is generated remained mysterious, with scientists searching for an elusive "high-energy intermediate" that would link oxidation and phosphorylation reactions.[120] This puzzle was solved byPeter D. Mitchell with the publication of thechemiosmotic theory in 1961.[121] At first, this proposal was highly controversial, but it was slowly accepted and Mitchell was awarded aNobel prize in 1978.[122][123] Subsequent research concentrated on purifying and characterizing the enzymes involved, with major contributions being made byDavid E. Green on the complexes of the electron-transport chain, as well asEfraim Racker on the ATP synthase.[124] A critical step towards solving the mechanism of the ATP synthase was provided byPaul D. Boyer, by his development in 1973 of the "binding change" mechanism, followed by his radical proposal of rotational catalysis in 1982.[76][125] More recent work has includedstructural studies on the enzymes involved in oxidative phosphorylation byJohn E. Walker, with Walker and Boyer being awarded a Nobel Prize in 1997.[126]
{{cite journal}}:ISBN / Date incompatibility (help)Metabolism map | ||
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 Majormetabolic pathways inmetro-style map. Click any text (name of pathway or metabolites) to link to the corresponding article. Single lines: pathways common to most lifeforms. Double lines: pathways not in humans (occurs in e.g. plants, fungi, prokaryotes).  Orange nodes:carbohydrate metabolism. Violet nodes:photosynthesis. Red nodes:cellular respiration. Pink nodes:cell signaling. Blue nodes:amino acid metabolism. Grey nodes:vitamin andcofactor metabolism. Brown nodes:nucleotide andprotein metabolism. Green nodes:lipid metabolism. | ||
