The below mentioned article provides a modern view on the electron transport system and oxidative phosphorylation in mitochondria.
Ever since the wide recognition of the fluid mosaic model of cell membranes, Mitchell’ chemiosomotic theory and more recently obtained knowledge about detailed structure and function of phosphorylating complexes (ATP synthase/ATPase) in mitochondria, our concept about electron transport chain and oxidative phosphorylation has also changed and become more clear.
It is now well established that the electron transport chain or system in mitochondria consists of four multi-protein complexes (called by Roman numerals I through IV) which are localised in the inner mitochondrial membrane and also ubiquinone (UQ or coenzyme Q) and cytochrome-c which are not tightly bound to membrane protein but act as mobile carriers between the complexes. (Fig. 16.6, 7, 9)
The composition of mitochondrial electron transport system is basically similar in most living organisms although there may be some minor variations in the nature of some of the components among groups of organisms. The electron transport system of animals and plants mitochondria is also similar; the main differences between the two are mentioned in the text that follows and are also apparent from the figures.
Consists of NADH-dehydrogenase (or NADH: Ubiquinone oxidoreductase) which contains a flavoprotein (FPint) FMN (Flavin Mono Nucleotide) and is associated with non-heme iron-sulphur (Fe-S) proteins.
This complex is responsible for passing electrons (also protons) from mitochondrial NADH/NADPH to Ubiquinone (UQ):
In plants (not animals), an additional external dehydrogenase complex is present which can oxidize cytosolic NADH (Fig. 16.7)
Consists of succinate dehydrogenase which contains a flavoprotein (FPs) called FAD (Flavin Adenine Dinucleotide) in its prosthetic group and is associated with nonheme iron- sulphur (Fe-S) proteins. This complex receives electrons (also protons) from succinic acid (which is oxidised in Krebs’ cycle to form fumaric acid vide reaction no. 17) and passes them to Ubiquinone (UQ).
Succinate + UQ → Fumarate + UQH2
Consists of Dihydroubiquinone (UQH2): cytochrome-C Oxido-reductase, two forms of cytochrome b (i.e., Cyt. b 562 and Cyt. b 566 in animal mitochondria and Cyt. b 566 and Cyt. b 560 or Cyt. b 557 and Cyt. b 560 in plant mitochondria), non-heme iron sulphur (Fe-S) proteins and cytochrome C1 (with E0‘ = + 0.22 V.).
In addition to these, this complex in plant mitochondria is also associated with a flavoprotein (FPha) which has a high (i.e. positive) E0‘ (+ 0.11 V) and a large absorbance change on redox change (Fig. 16.7). This complex receives electrons from UQH2 and passes them to cytochrome-C. The protons received from UQH2 are released out.
Consists of Cytochrome-C: Oxygen Oxidoreductase (Cytochrome Oxidase), Cytochrome-a and Cytochrome-a3. The enzyme of this complex contains copper (Cu) in the form of two copper centres CuA & CuB. This complex receives electrons from cytochrome-c and passes them to ½ O2. Two protons are needed and H2O molecule is formed (terminal oxidation).
(The organisation of these four complexes is quite specific in the inner mitochondrial membrane. NADH and FADH2 in complex I and II respectively are oxidised on the matrix side of the membrane. Ubiquinone can freely diffuse within the inner membrane. Cytochrome-c is loosely bound to the outer surface of the inner mitochondrial membrane i.e., towards outside of inner membrane and cytochrome b566 appears to face towards outside of inner membrane and cytochrome b560 is localised more towards inner side of the inner membrane.
In complex IV, one of the two copper centres of this complex CuA along with cytochrome a are localised towards outside of the inner membrane while CuB and cytochrome a3 are localised in the complex towards inner side (matrix side). In plants, the additional external NADH dehydrogenase complex is localised on the outer side (towards inter membrane space) of the inner mitochondrial membrane).
The transfer of electrons from reduced coenzyme NADH to oxygen via complexes I through IV is coupled to the synthesis of ATP from ADP and inorganic phosphate (Pi) which is called as oxidative phosphorylation. Experimental findings have confirmed that there are 3 sites of phosphorylation during mitochondrial electron transport, (i) during the transport of electrons from FPi of NADH dehydrogenise to UQ through Fe-S in complex I, (ii) from within the complex III to cytochrome-c and (iii) from cytochrome a to cytochrome a} in complex IV. Thus cytochrome a, in complex IV. Thus terminal oxidation of NADH results in the formation of 3ATP molecules while oxidation of FADH., results in production of only 2ATP molecules as it bypasses the first phosphorylation site (Fig. 16.6-7).
The widely accepted mechanism of mitochondrial synthesis of ATP is based on the chemiosmotic hypothesis first proposed by Mitchell in 1961 according to which asymmetric orientation of electron carriers within the inner mitochondrial membrane allows for the transfer of protons (H+) across the inner membrane during electron transport. It is now confirmed that mitochondrial electron transport is associated with translocation of protons from matrix to inter membrane space.
When electrons flow through complexes I, III or IV, these complexes act as proton pumps. They pump out protons across the inner membrane from matrix to inter membrane space. Because inner mitochondrial membrane is impermeable to protons (H+), a proton electrochemical gradient (∆~µH+) or a proton motive force is build up (H+ accumulate on the outside of the inner membrane in inter-membrane space which becomes acidic and positively charged while the inner side of the inner membrane i.e., matrix becomes alkaline and negatively charged). The free energy released during the electron transport is in-fact used to generate this proton motive force (proton electrochemical gradient).
(According to an estimate about 10 protons are pumped out across the inner mitochondrial membrane during electron transport by these complexes (4 protons by complex I, 4 protons by complex III and 2 protons by complex IV) for each pair of electrons that travels from NADH to ½O2.
Although the pumping of protons across the inner membrane can be explained by “redox loop” found in complex III, but the coupling of electron transport to proton translocation at complexes I & IV is not clearly understood. Oligomycin specifically blocks conduction of H+ through F0)
The free energy now stored in proton electro-chemical gradient or proton motive force can be used to carry chemical work i.e., synthesis of ATP from ADP + Pi. This is accomplished through phosphorylating complexes which are knob like structures situated on cristae in mitochondria (Fig. 16.8A) The phosphorylating complex or F0F1-ATP synthase (or F0F1-ATPase) which is sometimes called as complex V consists of two major components, a head piece or F1 and a basal part called F0.
The head piece F1 is a peripheral membrane protein complex projecting into matrix and consisting of 5 different subunits (3α, 3β subunits and one copy each of three other subunits called , δ and ɛ). It contains catalytic site for converting ADP + Pi into ATP or vice versa. The basal part or F0 is an integral membrane protein complex traversing across the inner mitochondrial membrane and consists of a cluster of many copies of at least 3 different small polypeptides called a, b and c which form a membrane channel for protons (Fig. 16.8B)
Based on biochemical and crystallographic studies, a model of F0F1 complex of mitochondria is shown in Fig. 16.10A. F0 complex consists of one a type, two b type (b2) and 10 c type (C10) polypeptide subunits. The two b subunits (b2) associate firmly with α and β subunits of F1 complex, holding them fixed relative to the membrane. The membrane embedded cylinder of c subunits (C10) is attached to the shaft made up of and ɛ subunits of F1 complex.
During mitochondrial electron transport, each time a proton electrochemical gradient (∆~µH+) or proton motive force is build up across the inner membrane due to pumping out of protons by complexes I, III or IV, protons from inter-membrane space diffuse down their electro-chemical gradient into matrix through channels formed by F0 components of phosphorylating complexes.
The passage of protons through these channels is coupled to the synthesis of ATP molecule from ADP + Pi by F1 component (i.e. ATP – synthase) simultaneously dissipating the proton electrochemical gradient. An overview of electron transport, proton translocation and oxidative phosphorylation is given in Fig. 16.9.
In recent years great advances have been made in our understanding of the synthesis of ATP in mitochondria especially due to research works carried out by Paul Boyer (1989, 93, 97) at the University of California, Los Angeles and John Walker and others (see Abrahams et al in references) in Cambridge, England who in 1994 determined high resolution X-ray structure of F1-ATPase from bovine heart mitochondria and confirmed the predictions of Paul Boyer’s binding change mechanism hypothesis and supported ‘rotational model’ for the catalytic mechanism of ATP synthesis (Fig. 16.10B) experimental.
According to this model, the movement of protons down their electrochemical gradient through channel in F0 complex drives the rotation of the entire F0 complex within the membrane. The ƴ subunit of the F1 complex which is attached to F0 complex also rotates within the catalytic complex like the shaft of a motor causing conformational changes in the catalytic complex for synthesis of ATP and its release from the catalytic sites situated on α and β subunits. For their contributions in elucidation of the mechanism of ATP synthesis, Paul Boyer and John walker were jointly awarded Nobel Prize in 1997 in Chemistry.
Oxidation of Extra-mitochondrial NADH (External NADH):
The NADH produced outside the mitochondria such as that produced during glycolysis in cytosol, cannot diffuse into the inner membrane of mitochondria which is impermeable to it and where electron transport chain is situated. The outer mitochondrial membrane is however, permeable to NADH. Therefore, the oxidation of this extra-mitochondrial NADH through electron transport chain in mitochondria poses certain problems.
In plant mitochondria there exists a provision to oxidise this NADH due to the presence of an external NADH dehydrogenase on the outer surface of the inner membrane of mitochondria. This enzyme contains flavoprotein (FPEXT) like the (FPINT) of internal NADH dehydrogenase but is not associated with Fe- S proteins.
The electrons from external NADH (which after diffusing through the outer mitochondrial membrane is now present in the space between outer and inner membranes of mitochondria) are transferred to Ubiquinone (UQ) directly bypassing the first site of phosphorylation (see Fig. 16.7).
In animal mitochondria (which differ from their counterparts in plants in not having an external NADH dehydrogenase), although external NADH cannot diffuse across the inner membrane, its reducing equivalents can diffuse into the latter by means of ‘shuttle mechanisms’. Two types of schuttle mechanisms are known in animals.
(1) Glycerophosphate schuttle:
This mechanism (Fig. 16.11) is present in insect flight muscle, brain, brown adipose tissue and white muscle and liver and involves the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate by cytosolic NADH in the presence of the enzyme glycerol-3-phosphate dehydrogenase.
Glycerol-3-phosphate enters into the mitochondrion where it is oxidised to form dihydroxyacetone phosphate again. This is coupled with the reduction of FAD into FADHr which enters into respiratory chain to yield 2ATP molecules. Dihydroxyacetone phosphate returns back to cytosol.
(2) Malate schuttle:
This mechanism (Fig. 16.12) occurs in animal tissues such as heart muscle. In this scheme, cytosolic NADH reduces oxaloacetate to malate which is carried across the inner mitochondrial membrane by a specific ketoglutaratemalate transporter. Inside the mitochondrion, malate is re-oxidised to oxaloacetate reducing NAD+ to NADH. This NADH can enter the respiratory chain to yield ATP molecules.
However, the complexity of this mechanism is due to the impermeability of inner mitochondrial membrane to oxaloacetate which must react with amino acid glutamate to form aspartate and α-ketoglutarate before transport through the inner mitochondrial membrane and reconstitution to oxaloacetate in cytosol. The inner membrane has glutamate-aspartate transporter.
In the operation of this schuttle, glutamate-asparate transporter also transfers a proton (H+) from cytosol into the mitochondrion (Fig. 16.12) thereby decreasing the proton motive force across the inner mitochondrial membrane. Therefore, oxidation of cytosolic NADH through this schuttle mechanism does not generate as much ATP as can be obtained from mitochondrial NADH (The number of ATP molecules produced by oxidation of cytosolic NADH through malate schuttle will not be exactly three but lesser than three).
Thus in both plants and animals (with glycero-phosphate schuttle) mitochondria, the terminal oxidation of external NADH bypasses the first phosphorylation site and hence, only two ATP molecules are produced per external NADH oxidised through mitochondrial electron transport chain. However, as mentioned earlier, in those animal tissues in which malate schuttle mechanism is present, the oxidation of external NADH will yield almost 3 ATP molecules.
Table 16.1 Simply shows balance sheet of ATP molecules produced and consumed during complete aerobic oxidation of hexose sugar in eukaryotes without taking into consideration the cost of their transport from mitochondrial matrix into the cytosol in exchange for ADP and Pi. This table is thus based on conventional view of only formation of 3ATP molecules by terminal oxidation of NADH and 2ATP molecules by terminal oxidation of FADH2.
However, taking into consideration the cost of transport of ATPs from matrix into the cytosol. This number will be 2.5 ATP for each of the NADH and 1.5 ATP for each of the FADH2 oxidised during electron transport system. Accordingly, there will be net cytosolic yield of 30 ATP molecules for complete aerobic oxidation of one molecule of glucose. But, in those animal tissue where malateschuttle mechanism is operative, the net yield will b6 32 ATP molecules (Table 16.1 A).
In those animal tissues where malate-schuttle mechanism is operative, 2 cytosolic NADH molecules will produce 2 × 2.5 = 5 ATP molecules and therefore, a net yield of 32 ATP molecules per glucose mol. oxidised. It is now generally agreed that during transfer of a pair of “electrons from NADH to O2 via electron transport chain, four protons (4 H+) each are pumped by complex I and complex III and two protons (2H+) are pumped by complex IV across the inner mitochondrial membrane from matrix into the inter-membrane space. When these protons move back into the matrix through channels formed by Fo components of ATP synthase complex, ATPs are synthesized.
ATP synthase utilizes three protons (3 H*) for the synthesis of 1 ATP molecule. The energetic cost of taking up one ADP and phosphate into the matrix and exporting 1 ATP into the cytosol is the movement of one proton (1 H+) from inter-membrane space into matrix. Thus total cost of synthesizing one ATP and its transport across inner membrane into the cytosol in exchange for ADP is four protons (4 H+). Specific transporters are present in inner membrane to facilitate this exchange.
Because, the rate of proton efflux and influx must balance, 2.5 ATP molecules (10/4) should be formed for each pair of electrons that are transferred from NADH to O2 via ETS. The P/O ratio (i.e., the number of ATP molecules synthesized per two electrons transferred down the respiratory chain to O2) thus is given by the ratio of proton stoichiometrics. If oxidation of succinate (or FADH2) extrudes six protons per pair of electrons (4 by complex III and 2 by complex IV), the P/O ratio for the substrate is 6/4 i.e., 1.5. These P/O ratios are in approximate agreement with the measured P/O ratios for the two substrates (Table 16.1 B).
It is noteworthy that each of the two ATP molecules formed by substrate level phosphorylation in TCA cycle (See reaction no. 16) either directly or through GTP, is in fact equivalent to about 3/4 molecule of ATP in the cytosol because exporting one ATP from mitochondria requires uptake of one proton (1/4 of the proton uptake required for synthesis and export of ATP by oxidative phosphorylation).
Therefore, net cytosolic yield of ATP from complete oxidation of 1 glucose molecule via glycolysis + TCA cycle will be 29.5 ATP molecules instead of 30 as discussed earlier. In those animal tissues where malate schuttle mechanism is operative, this figure will be 31 ATP molecules. It is because in such cases the oxidation of external NADH will not generate exactly 2.5 ATPs but lesser than it, the reason being that glutamate-aspartate transporter in malate schuttle mechanism also transfer a proton (H*) from cytosol into the mitochondrion.