8885ac19690-7503/1/0411:32 AM Page710mac76mac76:385 710 Chapter 19 Oxidative Phosphorylation and Photophosphorylation CADP B-ADP B-ATP ADP g-ATP B-empty FIGURE 19-23 Mitochondrial ATP synthase complex. (a) Structure of the F1 complex, deduced from crystallographic and biochemical studies In F1, three a and three B subunits are arranged like the seg. ments of an orange, with alternating a(shades of gray) and B(shades of purple) subunits around a central shaft, the y subunit (green).(b) Crystal structure of bovine F1(PDB ID 1 BMF), viewed from the side Two a subunits and one B subunit have been omitted to reveal the and ADP (yellow)on the B subunits. The 8 and e subunits are not shown here (c)F, viewed from above(that is, from the n side of the membrane showing the three and three a subunits and the central shaft (y sub- unit, green). Each B subunit, near its interface with the neighboring a subunit, has a nucleotide-binding site critical to the catalytic activity. The single y subunit associates primarily with one of the three aB John E. Walker pairs, forcing each of the three B subunits into slightly different con- formations, with different nucleotide-binding sites. In the crystalline enzyme, one subunit(B-ADP) has ADP (yellow) in its binding site, the When researchers crystallized the protein in the pres- next (B-ATP)has ATP (red), and the third (B-empty)has no bound nu- ence of ADP and App(Nd)p, a close structural analog cleotide. (d)Side view of the FFi structure. This is a composite, in of atP that cannot be hydrolyzed by the atPase activ. which the crystallographic coordinates of bovine mitochondrial F1 ity of F1, the binding site of one of the three B subunits mitochondrial Fo (shades of yellow and orange)(PDB ID 1Q01). Sub- was filled with App(NH)p, the second was filled with units a, b, 8, and e were not part of the crystal structure shown here (e)The FoF, structure, viewed end-on in the direction P side to n side NH The major structures visible in this cross section are the two trans- membrane helices of each of ten c subunits arranged in concentric circles. ()Diagram of the FF1 complex, deduced from biochemica CH2O-P--O- nd crystallographic studies. The two b subunits of F. associate firmly with the a and B subunits of F1, holding them fixed relative to the membrane. In Fo the membrane-embedded cylinder of c subunits is Nonhydrolyzable attached to the shaft made up of Fr subunits y and e As protons flow B-y bond through the membrane from the P side to the N side through For the OH OH cylinder and shaft rotate, and the B subunits of F, change conform App(NHp(B, y-imidoadenosine 5-triphosphate) ion as the y subunit associates with each in turn
When researchers crystallized the protein in the presence of ADP and App(NH)p, a close structural analog of ATP that cannot be hydrolyzed by the ATPase activity of F1, the binding site of one of the three subunits was filled with App(NH)p, the second was filled with 710 Chapter 19 Oxidative Phosphorylation and Photophosphorylation b-ATP b-ADP b-empty a-ADP a-empty a-ATP (c) ATP ADP (b) (a) FIGURE 19–23 Mitochondrial ATP synthase complex. (a) Structure of the F1 complex, deduced from crystallographic and biochemical studies. In F1, three and three subunits are arranged like the segments of an orange, with alternating (shades of gray) and (shades of purple) subunits around a central shaft, the subunit (green). (b) Crystal structure of bovine F1 (PDB ID 1BMF), viewed from the side. Two subunits and one subunit have been omitted to reveal the central shaft ( subunit) and the binding sites for ATP (red) and ADP (yellow) on the subunits. The and subunits are not shown here. (c) F1 viewed from above (that is, from the N side of the membrane), showing the three and three subunits and the central shaft ( subunit, green). Each subunit, near its interface with the neighboring subunit, has a nucleotide-binding site critical to the catalytic activity. The single subunit associates primarily with one of the three pairs, forcing each of the three subunits into slightly different conformations, with different nucleotide-binding sites. In the crystalline enzyme, one subunit (-ADP) has ADP (yellow) in its binding site, the next (-ATP) has ATP (red), and the third (-empty) has no bound nucleotide. (d) Side view of the FoF1 structure. This is a composite, in which the crystallographic coordinates of bovine mitochondrial F1 (shades of purple and gray) have been combined with those of yeast mitochondrial Fo (shades of yellow and orange) (PDB ID 1QO1). Subunits a, b, , and were not part of the crystal structure shown here. (e) The FoF1 structure, viewed end-on in the direction P side to N side. The major structures visible in this cross section are the two transmembrane helices of each of ten c subunits arranged in concentric circles. (f) Diagram of the FoF1 complex, deduced from biochemical and crystallographic studies. The two b subunits of Fo associate firmly with the and subunits of F1, holding them fixed relative to the membrane. In Fo, the membrane-embedded cylinder of c subunits is attached to the shaft made up of F1 subunits and . As protons flow through the membrane from the P side to the N side through Fo, the cylinder and shaft rotate, and the subunits of F1 change conformation as the subunit associates with each in turn. John E. Walker Nonhydrolyzable CH2O P P H O N O O O O H N N NH2 N N H OH OH H H P O O O O - bond App(NH)p (,-imidoadenosine 5-triphosphate) 8885d_c19_690-750 3/1/04 11:32 AM Page 710 mac76 mac76:385_reb:
8885ac19690-7503/1/0411:32 AM Page711mac76mac76:385 19.2 ATP Synthes 711 ADP ADP, and the third was empty. The corresponding B inner circle is made up of the amino-terminal helices of subunit conformations are designated B-ATP, B-ADP, each c subunit; the outer circle, about 55 A in diame- and B-empty(Fig. 19-23c). This difference in nucleo- ter, is made up of the carboxyl-terminal helices. The a tide binding among the three subunits is critical to the and y subunits of FI form a leg-and-foot that projects mechanism of the complex. from the bottom(membrane) side of F1 and stands The Fo complex making up the proton pore is firmly on the ring of c subunits. The schematic drawing composed of three subunits, a, b, and c, in the propor- in Figure 19-23f combines the structural information tion abe C10-12. Subunit c is a small (Mr 8,000), very from studies of bovine Fi and yeast FoFI hydrophobic polypeptide, consisting almost entirely of two transmembrane helices, with a small loop extend- Rotational Catalysis Is Key to the Binding-Change ng from the matrix side of the membrane. The crystal structure of the yeast FoFI, solved in 1999, shows the Mechanism for ATP Synthesis arrangement of the c subunits. The yeast complex has On the basis of detailed kinetic and binding studies of ten c subunits, each with two transmembrane helices the reactions catalyzed by FoFl, Paul Boyer proposed roughly perpendicular to the plane of the membrane and a rotational catalysis mechanism in which the three arranged in two concentric circles(Fig. 19-23d, e). The active sites of FI take turns catalyzing ATP synthesis
F1 Fo (d) ADP, and the third was empty. The corresponding subunit conformations are designated -ATP, -ADP, and -empty (Fig. 19–23c). This difference in nucleotide binding among the three subunits is critical to the mechanism of the complex. The Fo complex making up the proton pore is composed of three subunits, a, b, and c, in the proportion ab2c10–12. Subunit c is a small (Mr 8,000), very hydrophobic polypeptide, consisting almost entirely of two transmembrane helices, with a small loop extending from the matrix side of the membrane. The crystal structure of the yeast FoF1, solved in 1999, shows the arrangement of the c subunits. The yeast complex has ten c subunits, each with two transmembrane helices roughly perpendicular to the plane of the membrane and arranged in two concentric circles (Fig. 19–23d, e). The inner circle is made up of the amino-terminal helices of each c subunit; the outer circle, about 55 Å in diameter, is made up of the carboxyl-terminal helices. The and subunits of F1 form a leg-and-foot that projects from the bottom (membrane) side of F1 and stands firmly on the ring of c subunits. The schematic drawing in Figure 19–23f combines the structural information from studies of bovine F1 and yeast FoF1. Rotational Catalysis Is Key to the Binding-Change Mechanism for ATP Synthesis On the basis of detailed kinetic and binding studies of the reactions catalyzed by FoF1, Paul Boyer proposed a rotational catalysis mechanism in which the three active sites of F1 take turns catalyzing ATP synthesis 19.2 ATP Synthesis 711 (e) b2 c10 H+ a N side P side ADP + Pi ATP (f) 8885d_c19_690-750 3/1/04 11:32 AM Page 711 mac76 mac76:385_reb:
8885dc19690-7503/1/0411:32 AM Page712 6mac76:385 712 Chapter 19 Oxidative Phosphorylation and Photophosphorylation Fig. 19-24). A given B sub- this subunit again assumes the B-ADP form and binds nit starts in the B-ADP con- ADP and Pi formation. which binds ADP The conformational changes central to this mecha- and Pi from the surrounding nism are driven by the passage of protons through the medium. The subunit now Fo portion of ATP synthase. The streaming of protons changes conformation, assum- through the Fo "pore"causes the cylinder of c subunits ing the B-ATP form that and the attached y subunit to rotate about the long axis tightly binds and stabilizes of y, which is perpendicular to the plane of the mem- ATP, bringing about the ready brane. The y subunit passes through the center of the equilibration of ADP Pi with a B3 spheroid, which is held stationary relative to the Paul Boyer ATP on the enzyme surface. membrane surface by the be and 8 subunits (Fig Finally, the subunit changes to 3f). with each rotation of 120, y comes into con- the B-empty conformation, which has very low affinity tact with a different B subunit, and the contact forces for ATP, and the newly synthesized ATP leaves the en- that B subunit into the B-empty conformation zyme surface. Another round of catalysis begins when The three B subunits interact in such a way that when one assumes the B-empty conformation, its neigh bor to one side must assume the B-ADP form, and the other neighbor the B-ATP form. Thus one complete ro- tation of the y subunit causes each B subunit to cycle through all three of its possible conformations, and for ADP each rotation, three atP are synthesized and released from the One strong prediction of this binding-change model is that the y subunit should rotate in one direction when 3 Hp 3 H FoFI is synthesizing ATP and in the opposite direction when the enzyme is hydrolyzing ATP. This prediction was confirmed in elegant experiments in the laborato- ries of masasuke yoshida and kazuhiko kinosita jr The rotation of y in a single F1 molecule was observed mi- croscopically by attaching a long, thin, fluorescent actin polymer to y and watching it move relative to a B3 im mobilized on a microscope slide, as AtP was hydrolyzed. When the entire FoFI complex (not just F1 was used in a similar experiment, the entire ring of c subunits ro- tated with y(Fig. 19-25). The shaft"rotated in the pre- dicted direction through 360. The rotation was not smooth, but occurred in three discrete steps of 120.As calculated from the known rate of ATP hydrolysis by 3 H one Fi molecule and from the frictional drag on the long actin polymer, the efficiency of this mechanism in con- FIGURE 19-24 Binding-change model for ATP synthase. The Fr com- verting chemical energy into motion is close to 100%.It plex has three nonequivalent adenine nucleotide-binding sites, one for each pair of a and B subunits. At any given moment, one of these is, in Boyer's words, "a splendid molecular machine! sites is in the B-ATP conformation(which binds ATP tightly), a second is in the B-ADP (loose-binding) conformation, and a third is in the B- Chemiosmotic Coupling Allows Nonintegral empty (very-loose-binding) conformation. The proton-motive force Stoichiometries of O2 Consumption and ATP causes rotation of the central shaft-the y subunit, shown as a green Synthesis arrowhead-which comes into contact with each aB subunit pair in succession. This produces a cooperative conformational change Before the general acceptance of the chemiosmotic which the B-ATP site is converted to the B-empty conformation, and model for oxidative phosphorylation, the assumption ATP dissociates; the B-ADP site is converted to the B-ATP conforma. was that the overall reaction equation would take the tion, which promotes condensation of bound ADP P: to form ATE following form: and the B-empty site becomes a B-ADP site, which loosely binds ADP xADP+xP1+2O2+H++NADH→→ Pi entering from the solvent. This model, based on experimental xatP +Ho+ NAD findings, requires that at least two of the three catalytic sites altemate in activity; ATP cannot be released from one site unless and until ADP with the value of a-sometimes called the P/o ratio or and P are bound at the other the Pl2e ratio--always an integer. When intact mito-
(Fig. 19–24). A given subunit starts in the -ADP conformation, which binds ADP and Pi from the surrounding medium. The subunit now changes conformation, assuming the -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 -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 -ADP form and binds ADP and Pi . The conformational changes central to this mechanism are driven by the passage of protons through the Fo portion of ATP synthase. The streaming of protons through the Fo “pore” causes the cylinder of c subunits and the attached subunit to rotate about the long axis of , which is perpendicular to the plane of the membrane. The subunit passes through the center of the 33 spheroid, which is held stationary relative to the membrane surface by the b2 and subunits (Fig. 19–23f). With each rotation of 120 , comes into contact with a different subunit, and the contact forces that subunit into the -empty conformation. The three subunits interact in such a way that when one assumes the -empty conformation, its neighbor to one side must assume the -ADP form, and the other neighbor the -ATP form. Thus one complete rotation of the subunit causes each subunit to cycle through all three of its possible conformations, and for each rotation, three ATP are synthesized and released from the enzyme surface. One strong prediction of this binding-change model is that the subunit should rotate in one direction when FoF1 is synthesizing ATP and in the opposite direction when the enzyme is hydrolyzing ATP. This prediction was confirmed in elegant experiments in the laboratories of Masasuke Yoshida and Kazuhiko Kinosita, Jr. The rotation of in a single F1 molecule was observed microscopically by attaching a long, thin, fluorescent actin polymer to and watching it move relative to 33 immobilized on a microscope slide, as ATP was hydrolyzed. When the entire FoF1 complex (not just F1) was used in a similar experiment, the entire ring of c subunits rotated with (Fig. 19–25). The “shaft” rotated in the predicted direction through 360 . The rotation was not smooth, but occurred in three discrete steps of 120 . As calculated from the known rate of ATP hydrolysis by one F1 molecule and from the frictional drag on the long actin polymer, the efficiency of this mechanism in converting chemical energy into motion is close to 100%. It is, in Boyer’s words, “a splendid molecular machine!” Chemiosmotic Coupling Allows Nonintegral Stoichiometries of O2 Consumption and ATP Synthesis Before the general acceptance of the chemiosmotic model for oxidative phosphorylation, the assumption was that the overall reaction equation would take the following form: xADP xPi 1 2 O2 H NADH 88n xATP H2O NAD (19–11) with the value of x—sometimes called the P/O ratio or the P/2e ratio—always an integer. When intact mito- 712 Chapter 19 Oxidative Phosphorylation and Photophosphorylation ATP ATP ADP +Pi ATP ATP ADP +Pi ATP ATP ADP +Pi 3 HP + 3 HP + 3 HN + 3 HN + 3 HN + 3 HP + FIGURE 19–24 Binding-change model for ATP synthase. The F1 complex has three nonequivalent adenine nucleotide–binding sites, one for each pair of and subunits. At any given moment, one of these sites is in the -ATP conformation (which binds ATP tightly), a second is in the -ADP (loose-binding) conformation, and a third is in the - empty (very-loose-binding) conformation. The proton-motive force causes rotation of the central shaft—the subunit, shown as a green arrowhead—which comes into contact with each subunit pair in succession. This produces a cooperative conformational change in which the -ATP site is converted to the -empty conformation, and ATP dissociates; the -ADP site is converted to the -ATP conformation, which promotes condensation of bound ADP Pi to form ATP; and the -empty site becomes a -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. Paul Boyer 8885d_c19_690-750 3/1/04 11:32 AM Page 712 mac76 mac76:385_reb:
8885dc19690-7503/1/0411:32 AM Page713 19.2 ATP Synthesis FIGURE 19-25 Rotation of F. and y experimentally demonstrated. FI genetically engineered to contain a run of His residues adheres ightly to a microscope slide coated with a Ni complex; biotin is co- valently attached to a c subunit of Fo The protein avidin, which binds Actin filament biotin very tightly, is covalently attached to long filaments of actin la- beled with a fluorescent probe. Biotin-avidin binding now attache the actin filaments to the c subunit. When ATP is provided as sul Avidin strate for the ATPase activity of Fi, the labeled filament is seen to ate continuously in one direction, proving that the Fo cylinder of c subunits rotates. In another experiment, a fluorescent actin filament was attached directly to the y subunit. The series of fluorescence mi- crographs shows the position of the actin filament at intervals of 133 ms. Note that as the filament rotates, it makes a discrete jump about every eleventh frame. Presumably the cylinder and shaft move ADP +P ATP residues His residues PR Ni complex chondria are suspended in solution with an oxidizable and 6 for succinate. The most widely accepted experi substrate such as succinate or NADH and are provided mental value for number of protons required to drive with O2, ATP synthesis is readily measurable, as is the the synthesis of an ATP molecule is 4, of which l is used decrease in O2 Measurement of P/0, however, is com- in transporting Pi, ATP, and ADP across the mitochon licated by the fact that intact mitochondria consume drial membrane(see below). If 10 protons are pumped ATP in many reactions taking place in the matrix, and out per NADH and 4 must flow in to produce 1 ATP, the they consume O2 for purposes other than oxidative proton-based P/O ratio is 2.5 for NADH as the electron phosphorylation. Most experiments have yielded P/0 donor and 1.5(6/4) for succinate. We use the P/O val- (ATP to O2) ratios of between 2 and 3 when NADH ues of 2.5 and 1.5 throughout this book, but the values was the electron donor and between I and 2 when suc- 3.0 and 2.0 are still common in the biochemical litera cinate was the donor. Given the assumption that p/o ture. The final word on proton stoichiometry will prob- should have an integral value, most experimenters ably not be written until we know the full details of the agreed that the P/O ratios must be 3 for NADH and 2 FoFI reaction mechanism. for succinate, and for years those values have appeared in research papers and textbooks. with introduction of the chemiosmotic paradigm for The Proton-Motive Force Energizes Active Transport coupling ATP synthesis to electron transfer, there was Although the primary role of the proton gradient in mi- no theoretical requirement for P/0 to be integral. The tochondria is to furnish energy for the synthesis of ATP, relevant questions about stoichiometry became, how the proton-motive force also drives several transport many protons are pumped outward by electron transfer processes essential to oxidative phosphorylation. The from one NADH to O2, and how many protons must flow inner mitochondrial membrane is generally imperme- inward through the Fo FI complex to drive the synthe- able to charged species, but two specific systems trans- sis of one ATP? The measurement of proton fluxes port ADP and Pi into the matrix and ATP out to the cy technically complicated; the investigator must take into toso (Fig. 19-26) account the buffering capacity of mitochondria, non- The adenine nucleotide translocase, integral to productive leakage of protons across the inner mem- the inner membrane, binds ADP in the intermembrane brane, and use of the proton gradient for functions other space and transports it into the matrix in exchange for than ATP synthesis, such as driving the transport of sub- an ATP- molecule simultaneously transported outward strates across the inner mitochondrial membrane (de- (see Fig 13-1 for the ionic forms of ATP and ADP). Be scribed below). The consensus values for number of pro- cause this antiporter moves four negative charges out tons pumped out per pair of electrons are 10 for NADH for every three moved in, its activity is favored by the
ADP + Pi ATP Ni complex His residues His residues Avidin Fo F1 a b c Actin filament chondria are suspended in solution with an oxidizable substrate such as succinate or NADH and are provided with O2, ATP synthesis is readily measurable, as is the decrease in O2. Measurement of P/O, however, is complicated by the fact that intact mitochondria consume ATP in many reactions taking place in the matrix, and they consume O2 for purposes other than oxidative phosphorylation. Most experiments have yielded P/O (ATP to 1 2 O2) ratios of between 2 and 3 when NADH was the electron donor, and between 1 and 2 when succinate was the donor. Given the assumption that P/O should have an integral value, most experimenters agreed that the P/O ratios must be 3 for NADH and 2 for succinate, and for years those values have appeared in research papers and textbooks. With introduction of the chemiosmotic paradigm for coupling ATP synthesis to electron transfer, there was no theoretical requirement for P/O to be integral. The relevant questions about stoichiometry became, how many protons are pumped outward by electron transfer from one NADH to O2, and how many protons must flow inward through the FoF1 complex to drive the synthesis of one ATP? The measurement of proton fluxes is technically complicated; the investigator must take into account the buffering capacity of mitochondria, nonproductive leakage of protons across the inner membrane, and use of the proton gradient for functions other than ATP synthesis, such as driving the transport of substrates across the inner mitochondrial membrane (described below). The consensus values for number of protons pumped out per pair of electrons are 10 for NADH and 6 for succinate. The most widely accepted experimental value for number of protons required to drive the synthesis of an ATP molecule is 4, of which 1 is used in transporting Pi , ATP, and ADP across the mitochondrial membrane (see below). If 10 protons are pumped out per NADH and 4 must flow in to produce 1 ATP, the proton-based P/O ratio is 2.5 for NADH as the electron donor and 1.5 (6/4) for succinate. We use the P/O values of 2.5 and 1.5 throughout this book, but the values 3.0 and 2.0 are still common in the biochemical literature. The final word on proton stoichiometry will probably not be written until we know the full details of the FoF1 reaction mechanism. The Proton-Motive Force Energizes Active Transport Although the primary role of the proton gradient in mitochondria is to furnish energy for the synthesis of ATP, the proton-motive force also drives several transport processes essential to oxidative phosphorylation. The inner mitochondrial membrane is generally impermeable to charged species, but two specific systems transport ADP and Pi into the matrix and ATP out to the cytosol (Fig. 19–26). The adenine nucleotide translocase, integral to the inner membrane, binds ADP3 in the intermembrane space and transports it into the matrix in exchange for an ATP4 molecule simultaneously transported outward (see Fig. 13–1 for the ionic forms of ATP and ADP). Because this antiporter moves four negative charges out for every three moved in, its activity is favored by the 19.2 ATP Synthesis 713 FIGURE 19–25 Rotation of Fo and experimentally demonstrated. F1 genetically engineered to contain a run of His residues adheres tightly to a microscope slide coated with a Ni complex; biotin is covalently attached to a c subunit of Fo. The protein avidin, which binds biotin very tightly, is covalently attached to long filaments of actin labeled with a fluorescent probe. Biotin-avidin binding now attaches the actin filaments to the c subunit. When ATP is provided as substrate for the ATPase activity of F1, the labeled filament is seen to rotate continuously in one direction, proving that the Fo cylinder of c subunits rotates. In another experiment, a fluorescent actin filament was attached directly to the subunit. The series of fluorescence micrographs shows the position of the actin filament at intervals of 133 ms. Note that as the filament rotates, it makes a discrete jump about every eleventh frame. Presumably the cylinder and shaft move as one unit. 8885d_c19_690-750 3/1/04 11:32 AM Page 713 mac76 mac76:385_reb:
8885dc19690-7503/1/0411:32 AM Page714 6mac76:385 714 Chapter 19 Oxidative Phosphorylation and Photophosphorylation Intermembrane Matrix mitochondria by gentle dissection with detergents, sug- gesting that the functions of these three proteins are ery tightly integrated ATP4- Shuttle Systems Indirectly Convey Cytosolic NADH into Mitochondria for Oxidation ADI The NAdh dehydrogenase of the inner mitochondrial membrane of animal cells can accept electrons only from ATP NAdH in the matrix. Given that the inner membrane is not permeable to NADH, how can the Nadh generated by glycolysis in the cytosol be reoxidized to nAd by O2 via the respiratory chain? Special shuttle systems H2POz carry reducing equivalents from cytosolic NADH into Phosphate hpoz mitochondria by an indirect route. The most active symporter)H+ NADH Shuttle, which functions in liver, kidney, and heart mitochondria, is the malate-aspartate shuttle (Fig. 19-20. The reducing equivalents of cytosolic NADH are first transferred to cytosolic oxaloacetate to yield malate, catalyzed by cytosolic malate dehydroge- FIGURE 19-26 Adenine nucleotide and phosphate translocases. nase. The malate thus formed passes through the inner Transport systems of the inner mitochondrial membrane carry ADI membrane via the malate-ar-ketoglutarate transporter and P, into the matrix and newly synthesized ATP into the cytosol. within the matrix the reducing equivalents are passed The adenine nucleotide translocase is an antiporter; the same protein to nad by the action of matrix malate dehydrogenase moves ADP into the matrix and ATP out. The effect of replacing ATP4 forming NADH; this NADH can pass electrons directly th ADP- is the net efflux of one negative charge, which is favored to the respiratory chain. About 2.5 molecules of ATP are by the charge difference across the inner membrane (outside positive). generated as this pair of electrons passes to O2. Cy At pH 7, P: is present as both HPOZ- and H, POA; the phosphate tosolic oxaloacetate must be regenerated by transami- translocase is specific for H2PO4. There is no net flow of charge dur- nation reactions and the activity of membrane trans- ing symport of H2 PO4 and H+, but the relatively low proton con- porters to start another cycle of the shuttle centration in the matrix favors the inward movement of H+. Thus the Skeletal muscle and brain use a different nadh proton-motive force is responsible both for providing the energy for shuttle, the glycerol 3-phosphate shuttle (Fig ATP synthesis and for transporting substrates (ADP and Pi) in and prod- 19-28). It differs from the malate-aspartate shuttle in uct(ATP)out of the mitochondrial matrix. All three of these transport that it delivers the reducing equivalents from NAdh to systems can be isolated as a single membrane-bound complex (ATP ubiquinone and thus into Complex Ill, not Complex I (Fig. 19-8), providing only enough energy to synthesize 1.5 ATP molecules air of electrons The mitochondria of plants have an externally ori- ransmembrane electrochemical gradient, which gives ented NADH dehydrogenase that can transfer electrons the matrix a net negative charge; the proton-motive directly from cytosolic NADH into the respiratory chain force drives ATP-ADP exchange. Adenine nucleotide at the level of ubiquinone. Because this pathway by- translocase is specifically inhibited by atractyloside, a passes the NADH dehydrogenase of Complex I and the toxic glycoside formed by a species of thistle. If the associated proton movement, the yield of Arp from cy- transport of ADP into and ATP out of mitochondria tosolic NADH is less than that from NAdh generated in inhibited, cytosolic ATP cannot be regenerated from the matrix(Box 19-1) ADP, explaining the toxicity of atractyloside A second membrane transport system essential SUMMARY 19.2 ATP Synthesis oxidative phosphorylation is the phosphate translo case, which promotes symport of one H2 POA and one I The flow of electrons through Complexes L, Il into the matrix. This transport process, too, is fa and Iv results in pumping of protons across the vored by the transmembrane proton gradient (Fig. inner mitochondrial membrane, making the 19-26). Notice that the process requires movement of matrix alkaline relative to the intermembrane one proton from the p to the n side of the inner mem- space. This proton gradient provides the energy brane, consuming some of the energy of electron trans- (in the form of the proton-motive force)for fer. A complex of the ATP synthase and both translo ATP synthesis from ADP and Pi by ATP synthase cases, the AtP synthasome, can be isolated from (FoFI complex) in the inner membrane
transmembrane electrochemical gradient, which gives the matrix a net negative charge; the proton-motive force drives ATP-ADP exchange. Adenine nucleotide translocase is specifically inhibited by atractyloside, a toxic glycoside formed by a species of thistle. If the transport of ADP into and ATP out of mitochondria is inhibited, cytosolic ATP cannot be regenerated from ADP, explaining the toxicity of atractyloside. A second membrane transport system essential to oxidative phosphorylation is the phosphate translocase, which promotes symport of one H2PO4 and one H into the matrix. This transport process, too, is favored by the transmembrane proton gradient (Fig. 19–26). Notice that the process requires movement of one proton from the P to the N side of the inner membrane, consuming some of the energy of electron transfer. A complex of the ATP synthase and both translocases, the ATP synthasome, can be isolated from mitochondria by gentle dissection with detergents, suggesting that the functions of these three proteins are very tightly integrated. Shuttle Systems Indirectly Convey Cytosolic NADH into Mitochondria for Oxidation The NADH dehydrogenase of the inner mitochondrial membrane of animal cells can accept electrons only from NADH in the matrix. Given that the inner membrane is not permeable to NADH, how can the NADH generated by glycolysis in the cytosol be reoxidized to NAD by O2 via the respiratory chain? Special shuttle systems carry reducing equivalents from cytosolic NADH into mitochondria by an indirect route. The most active NADH shuttle, which functions in liver, kidney, and heart mitochondria, is the malate-aspartate shuttle (Fig. 19–27). The reducing equivalents of cytosolic NADH are first transferred to cytosolic oxaloacetate to yield malate, catalyzed by cytosolic malate dehydrogenase. The malate thus formed passes through the inner membrane via the malate–-ketoglutarate transporter. Within the matrix the reducing equivalents are passed to NAD by the action of matrix malate dehydrogenase, forming NADH; this NADH can pass electrons directly to the respiratory chain. About 2.5 molecules of ATP are generated as this pair of electrons passes to O2. Cytosolic oxaloacetate must be regenerated by transamination reactions and the activity of membrane transporters to start another cycle of the shuttle. Skeletal muscle and brain use a different NADH shuttle, the glycerol 3-phosphate shuttle (Fig. 19–28). It differs from the malate-aspartate shuttle in that it delivers the reducing equivalents from NADH to ubiquinone and thus into Complex III, not Complex I (Fig. 19–8), providing only enough energy to synthesize 1.5 ATP molecules per pair of electrons. The mitochondria of plants have an externally oriented NADH dehydrogenase that can transfer electrons directly from cytosolic NADH into the respiratory chain at the level of ubiquinone. Because this pathway bypasses the NADH dehydrogenase of Complex I and the associated proton movement, the yield of ATP from cytosolic NADH is less than that from NADH generated in the matrix (Box 19–1). SUMMARY 19.2 ATP Synthesis ■ 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. 714 Chapter 19 Oxidative Phosphorylation and Photophosphorylation Intermembrane space Matrix Adenine nucleotide translocase (antiporter) ATP synthase Phosphate translocase (symporter) ATP4 H H2PO4 H ATP4 ADP3 H H2PO4 H ADP3 FIGURE 19–26 Adenine nucleotide and phosphate translocases. Transport systems of the inner mitochondrial membrane carry ADP and Pi into the matrix and newly synthesized ATP into the cytosol. The adenine nucleotide translocase is an antiporter; the same protein moves ADP into the matrix and ATP out. The effect of replacing ATP4 with ADP3 is the net efflux of one negative charge, which is favored by the charge difference across the inner membrane (outside positive). At pH 7, Pi is present as both HPO4 2 and H2PO4 ; the phosphate translocase is specific for H2PO4 . There is no net flow of charge during symport of H2PO4 and H, but the relatively low proton concentration in the matrix favors the inward movement of H. Thus the proton-motive force is responsible both for providing the energy for ATP synthesis and for transporting substrates (ADP and Pi) in and product (ATP) out of the mitochondrial matrix. All three of these transport systems can be isolated as a single membrane-bound complex (ATP synthasome). 8885d_c19_690-750 3/1/04 11:32 AM Page 714 mac76 mac76:385_reb: