19.3 Regulation of Oxidative Phosphorylation in Chapter 19 Oxidative Phosphorylation

19.3 Regulation of Oxidative Phosphorylation

Oxidative phosphorylation produces most of the ATP made in aerobic cells. Complete oxidation of a molecule of glucose to ${CO}_{2}$ $CO Subscript 2$ yields 30 or 32 ATP (Table 19-5). By comparison, glycolysis under anaerobic conditions (lactate fermentation) yields only 2 ATP per glucose. Clearly, the evolution of oxidative phosphorylation provided a tremendous increase in the energy efficiency of catabolism. Complete oxidation to ${CO}_{2}$ $CO Subscript 2$ of the coenzyme A derivative of palmitate (16:0), which also occurs in the mitochondrial matrix, yields 108 ATP per palmitoyl-CoA (see Table 17-1). A similar calculation can be made for the ATP yield from oxidation of each of the amino acids (Chapter 18). Aerobic oxidative pathways that result in electron transfer to $O_{2}$ $upper O Subscript 2$ accompanied by oxidative phosphorylation therefore account for the vast majority of the ATP produced in catabolism, so the regulation of ATP production by oxidative phosphorylation to match the cell’s fluctuating needs for ATP is absolutely essential.

TABLE 19-5 ATP Yield from Complete Oxidation of Glucose
Process	Direct product	Final ATP
Glycolysis	2 NADH (cytosolic) 2 ATP	3 or 5^a 2
Pyruvate oxidation (two per glucose)	2 NADH (mitochondrial matrix)	5
Acetyl-CoA oxidation in citric acid cycle (two per glucose)	6 NADH (mitochondrial matrix) $2 {FADH}_{2}$ $2 FADH Subscript 2 Baseline$ 2 ATP or 2 GTP	15 3 2
Total yield per glucose		30 or 32
^aIf the malate/aspartate shuttle is used to transfer reducing equivalents into the mitochondrion, the yield is 5 ATP. If the glycerol 3-phosphate shuttle is used, the yield is 3 ATP.

Oxidative Phosphorylation Is Regulated by Cellular Energy Needs

The rate of respiration ( $O_{2}$ $upper O Subscript 2$ consumption) in mitochondria is generally limited by the availability of ADP as a substrate for phosphorylation. Dependence of the rate of $O_{2}$ $upper O Subscript 2$ consumption on the availability of the $P_{i}$ $upper P Subscript i$ acceptor, ADP (Fig. 19-20b), the acceptor control of respiration, can be remarkable. In some animal tissues, the acceptor control ratio, the ratio of the maximal rate of ADP-induced $O_{2}$ $upper O Subscript 2$ consumption to the basal rate in the absence of ADP, is at least 10.

The intracellular concentration of ADP is one measure of the energy status of cells. Another, related measure is the mass-action ratio of the ATP-ADP system, $[ATP] / ([ADP] [P_{i}])$ $left-bracket ATP right-bracket slash left-parenthesis left-bracket ADP right-bracket left-bracket upper P Subscript i Baseline right-bracket right-parenthesis$ . Usually this ratio is very high, so the ATP-ADP system is almost fully phosphorylated. When the rate of some energy-requiring process (protein synthesis, for example) increases, the rate of breakdown of ATP to ADP and $P_{i}$ $upper P Subscript i$ increases, lowering the mass-action ratio. With more ADP available for oxidative phosphorylation, the rate of respiration increases, causing regeneration of ATP. This continues until the mass-action ratio returns to its normal high level, at which point respiration slows again. The rate of oxidation of cellular fuels is regulated with such sensitivity and precision that the $[ATP] / ([ADP] [P_{i}])$ $left-bracket ATP right-bracket slash left-parenthesis left-bracket ADP right-bracket left-bracket upper P Subscript i Baseline right-bracket right-parenthesis$ ratio fluctuates only slightly in most tissues, even during extreme variations in energy demand. In short, ATP is formed only as fast as it is used in energy-requiring cellular activities.

An Inhibitory Protein Prevents ATP Hydrolysis during Hypoxia

We have already encountered ATP synthase as an ATP-driven proton pump (see Fig. 11-40), catalyzing the reverse of ATP synthesis under some experimental conditions. When a cell is hypoxic (deprived of oxygen), as in a heart attack or a stroke, electron transfer to oxygen slows, and so does the pumping of protons. The proton-motive force soon collapses. Under these conditions, the ATP synthase could operate in reverse, hydrolyzing ATP made by glycolysis to pump protons outward and causing a disastrous drop in ATP levels. This is prevented by a small (84 amino acids) protein inhibitor, ${IF}_{1}$ $IF Subscript 1$ . ${IF}_{1}$ $IF Subscript 1$ simultaneously binds to two ATP synthase molecules, inhibiting the enzyme’s activity in both directions (Fig. 19-33). ${IF}_{1}$ $IF Subscript 1$ is inhibitory only in its dimeric form, which is favored at pH lower than 6.5. In a cell starved for oxygen, the main source of ATP becomes glycolysis, and the pyruvic or lactic acid thus formed lowers the pH in the cytosol and the mitochondrial matrix. This favors ${IF}_{1}$ $IF Subscript 1$ dimerization, leading to inhibition of ATP synthase and thereby preventing any wasteful hydrolysis of ATP. When aerobic metabolism resumes, production of pyruvic acid slows, the pH of the cytosol rises, the ${IF}_{1}$ $IF Subscript 1$ dimer is destabilized, and the inhibition of ATP synthase is lifted. ${IF}_{1}$ $IF Subscript 1$ is an intrinsically disordered protein (p. 117); it acquires a favored conformation only on interaction with ATP synthase. In many tumors and cancer cell lines, which rely more heavily on glycolysis for ATP generation, ${IF}_{1}$ $IF Subscript 1$ is expressed at unusually high levels.

A figure shows the structure of bovine F subscript 1 end subscript-A T P ase in complex with its regulatory protein, I F subscript 1 end subscript. — FIGURE 19-33 Structure of bovine $F_{1}$ $upper F Subscript 1$ -ATPase in a complex with its regulatory protein ${IF}_{1}$ $IF Subscript 1$ . Two $F_{1}$ $upper F Subscript 1$ molecules are viewed here from the n side, as in Figure 19-25b. The inhibitor ${IF}_{1}$ $IF Subscript 1$ (red) binds to the $α β$ $alpha beta$ interface of the subunits in the diphosphate (ADP) conformation (α-ADP and β-ADP), freezing the two $F_{1}$ $upper F Subscript 1$ complexes and thereby blocking ATP hydrolysis (and synthesis). (Parts of the ${IF}_{1} α$ $IF Subscript 1 Baseline alpha$ -helices that link the two $F_{1}$ $upper F Subscript 1$ molecules failed to resolve in the crystals of $F_{1}$ $upper F Subscript 1$ and are modeled based on the crystal structure of isolated ${IF}_{1}$ $IF Subscript 1$ .) [Data from PDB ID 1OHH, E. Cabezon et al., *Nat. Struct. Biol.* 10:744, 2003.]

FIGURE 19-33 Structure of bovine $F_{1}$ $upper F Subscript 1$ -ATPase in a complex with its regulatory protein ${IF}_{1}$ $IF Subscript 1$ . Two $F_{1}$ $upper F Subscript 1$ molecules are viewed here from the n side, as in Figure 19-25b. The inhibitor ${IF}_{1}$ $IF Subscript 1$ (red) binds to the $α β$ $alpha beta$ interface of the subunits in the diphosphate (ADP) conformation (α-ADP and β-ADP), freezing the two $F_{1}$ $upper F Subscript 1$ complexes and thereby blocking ATP hydrolysis (and synthesis). (Parts of the ${IF}_{1} α$ $IF Subscript 1 Baseline alpha$ -helices that link the two $F_{1}$ $upper F Subscript 1$ molecules failed to resolve in the crystals of $F_{1}$ $upper F Subscript 1$ and are modeled based on the crystal structure of isolated ${IF}_{1}$ $IF Subscript 1$ .) [Data from PDB ID 1OHH, E. Cabezon et al., *Nat. Struct. Biol.* 10:744, 2003.]

Two roughly circular structures are shown. The left-hand structure has a green piece in the center that is roughly rectangular but bumpy with rounded sides. Six pieces surround it in a circle, each narrower toward the center and wider toward the outside. There is a purple piece labeled beta at the upper left, a gray piece labeled alpha at the upper right, a purple piece labeled beta at the right, a gray piece labeled alpha at the lower right, a purple piece labeled beta at the lower left, and a gray piece labeled alpha at the left side. A similar circular structure is to its right, but flipped so that the bottom is now on the top. A red helix begins at the green central piece of the left-hand structure and extends across the purple piece on the right side, then becomes orange as it passes across the open region between the two circular structures and crosses an orange end of a similar helix that extends right, becomes red as it passes the left-hand purple part of the right-hand structure, and ends at the green center of the right-hand structure. Each of these long red and orange helices is labeled I F subscript 1 end subscript inhibitor.

Hypoxia Leads to ROS Production and Several Adaptive Responses

In hypoxic cells there is an imbalance between the input of electrons from fuel oxidation in the mitochondrial matrix and transfer of electrons to molecular oxygen, leading to increased formation of reactive oxygen species. In addition to the glutathione peroxidase system (Fig. 19-18), cells have two other lines of defense against ROS (Fig. 19-34). One is regulation of pyruvate dehydrogenase (PDH), the enzyme that delivers acetyl-CoA to the citric acid cycle (Chapter 16). Under hypoxic conditions, PDH kinase phosphorylates mitochondrial PDH, inactivating it and slowing the delivery of ${FADH}_{2}$ $FADH Subscript 2$ and NADH from the citric acid cycle to the respiratory chain. A second means of preventing ROS formation is the replacement of one subunit of Complex IV, known as COX4-1, with another subunit, COX4-2, that is better suited to hypoxic conditions. With COX4-1, the catalytic properties of Complex IV are optimal for respiration at normal oxygen concentrations; with COX4-2, Complex IV is optimized for operation under hypoxic conditions.

A figure shows the regulation of gene expression by hypoxia-inducible factor (H I F – 1) to reduce R O S formation. — FIGURE 19-34 Regulation of gene expression by hypoxia-inducible factor (HIF-1) to reduce ROS formation. Under conditions of low oxygen (hypoxia), HIF-1 is synthesized in greater amounts and acts as a transcription factor, increasing synthesis of the glucose transporter, glycolytic enzymes, pyruvate dehydrogenase kinase (PDH kinase), lactate dehydrogenase, a protease that degrades the cytochrome oxidase subunit COX4-1, and cytochrome oxidase subunit COX4-2. These changes counter the formation of ROS by decreasing the supply of NADH and ${FADH}_{2}$ $FADH Subscript 2$ and making cytochrome oxidase of Complex IV more effective. [Information from D. A. Harris, *Bioenergetics at a Glance,* p. 36, Blackwell Science, 1995.]

FIGURE 19-34 Regulation of gene expression by hypoxia-inducible factor (HIF-1) to reduce ROS formation. Under conditions of low oxygen (hypoxia), HIF-1 is synthesized in greater amounts and acts as a transcription factor, increasing synthesis of the glucose transporter, glycolytic enzymes, pyruvate dehydrogenase kinase (PDH kinase), lactate dehydrogenase, a protease that degrades the cytochrome oxidase subunit COX4-1, and cytochrome oxidase subunit COX4-2. These changes counter the formation of ROS by decreasing the supply of NADH and ${FADH}_{2}$ $FADH Subscript 2$ and making cytochrome oxidase of Complex IV more effective. [Information from D. A. Harris, *Bioenergetics at a Glance,* p. 36, Blackwell Science, 1995.]

A key indicates that a green arrow means increased transcription, a dashed arrow means decreased flow through pathway, and a thick gray arrow means increased flow through pathway. The figure begins with bolded text at the top reading, hypoxia (low p O 2). An arrow pointing down is accompanied by a gray box reading, increase transcription of H I F – 1. The arrow points to H I F – 1 with an upward-pointing green arrow to its left. A gray box below reads, H I F – 1 increases transcription of other enzymes and proteins. Two arrows point away from H I F – 1. The left-hand arrow points left and then down to glucose transporter next to an upward-pointing green arrow to its left. To the right, a thick gray arrow points down to glucose. A thick arrow points down from glucose to pyruvate with an arrow curving away to show loss of A T P. Accompanying text reads, upward-pointing green arrow symbol glycolytic enzymes. An accompanying gray box reads, A T P production by glycolysis increases. A thick gray arrow points left from glucose to lactate beneath text reading, upward-pointing green arrow lactate blue highlighted dehydrogenase. A dashed arrow labeled blue highlighted pyruvate dehydrogenase (P D H) points down from pyruvate to acetyl Co A at the start of a dashed clockwise arrow that ends back at acetyl Co A and is labeled citric acid cycle. A red circle containing a red “X” is next to the dashed arrow labeled pyruvate dehydrogenase (P D H) and this circle is indicated by a dashed line from upward-pointing green arrow blue highlighted P D H kinase. A dashed arrow points form the citric acid cycle to N A D H, F A D H 2 above a gray box reading, electron flow from N A D H and F A D H 2 to respiratory chain decreases. A dashed arrow labeled respiratory chain points from N A D H, F A D H 2 to complex Roman numeral 4 accompanied by a dashed arrow from O 2 to O 2 with a dot to the upper left and a negative sign, O H with a dot on O and a gray box below reading, R O S production is reduced. A curved arrow from O 2 to H 2 O passes through complex Roman numeral 4. The right-hand arrow from upward-pointing green arrow symbol H I F – 1 points right and then down to upward-pointing green arrow symbol protease with an arrow pointing right to degrades C O X 4 – 1 subunit above upward-pointing green arrow symbol C O X 4 – 2 subunit with an arrow pointing right to text reading, replaces C O X 4 – 1. A gray box below reads, Complex Roman numeral 4 properties are adapted to low p O 2.

The changes in PDH activity and the COX4-2 content of Complex IV are both mediated by HIF-1, the hypoxia-inducible factor. HIF-1 (another intrinsically disordered protein) accumulates in hypoxic cells and, acting as a transcription factor, triggers increased synthesis of PDH kinase, COX4-2, and a protease that degrades COX4-1. HIF-1 is a master regulator of $O_{2}$ $upper O Subscript 2$ homeostasis. Recall that it also mediates the changes in glucose transport and glycolytic enzymes that produce the Warburg effect, the dependence on glycolysis (not mitochondrial respiration) for ATP production, even in the presence of sufficient oxygen (see Box 14-1).

When these mechanisms for dealing with ROS are insufficient, either due to genetic mutation affecting one of the protective proteins or under conditions of very high rates of ROS production, mitochondrial function is compromised. Mitochondrial damage is thought to be involved in aging, heart failure, certain rare cases of diabetes (described below), and several maternally inherited genetic diseases that affect the nervous system.

ATP-Producing Pathways Are Coordinately Regulated

The major catabolic pathways have overlapping and concerted regulatory mechanisms that allow them to function together in an economical and self-regulating manner to produce ATP and biosynthetic precursors. The relative concentrations of ATP and ADP control not only the rates of electron transfer and oxidative phosphorylation but also the rates of the citric acid cycle, pyruvate oxidation, and glycolysis (Fig. 19-35). Whenever ATP consumption increases, the rate of electron transfer and oxidative phosphorylation increases. Simultaneously, the rate of pyruvate oxidation via the citric acid cycle increases, increasing the flow of electrons into the respiratory chain. These events, in turn, can evoke an increased rate of glycolysis, increasing the rate of pyruvate formation. When conversion of ADP to ATP lowers the ADP concentration, acceptor control slows electron transfer and thus oxidative phosphorylation. Glycolysis and the citric acid cycle are also slowed, because ATP is an allosteric inhibitor of the glycolytic enzyme phosphofructokinase-1 (see Fig. 14-23) and of pyruvate dehydrogenase (see Fig. 16-18).

A figure shows the regulation of A T P-producing pathways. — FIGURE 19-35 Regulation of ATP-producing pathways. This diagram shows the coordinated regulation of glycolysis, pyruvate oxidation, the citric acid cycle, and oxidative phosphorylation by the relative concentrations of ATP, ADP, and AMP, and by NADH. High [ATP] (or low [ADP] and [AMP]) produces low rates of glycolysis, pyruvate oxidation, acetate oxidation via the citric acid cycle, and oxidative phosphorylation. All four pathways are accelerated when the use of ATP and the formation of ADP, AMP, and $P_{i}$ $upper P Subscript i$ increase. The ability of citrate to inhibit both glycolysis and the citric acid cycle reinforces the action of the adenine nucleotide system. In addition, increased [NADH] and [acetyl-CoA] also inhibit the oxidation of pyruvate to acetyl-CoA, and a high $[NADH] / [{NAD}^{+}]$ $left-bracket NADH right-bracket slash left-bracket NAD Superscript plus Baseline right-bracket$ ratio inhibits the dehydrogenase reactions of the citric acid cycle (see Fig. 16-18).

FIGURE 19-35 Regulation of ATP-producing pathways. This diagram shows the coordinated regulation of glycolysis, pyruvate oxidation, the citric acid cycle, and oxidative phosphorylation by the relative concentrations of ATP, ADP, and AMP, and by NADH. High [ATP] (or low [ADP] and [AMP]) produces low rates of glycolysis, pyruvate oxidation, acetate oxidation via the citric acid cycle, and oxidative phosphorylation. All four pathways are accelerated when the use of ATP and the formation of ADP, AMP, and $P_{i}$ $upper P Subscript i$ increase. The ability of citrate to inhibit both glycolysis and the citric acid cycle reinforces the action of the adenine nucleotide system. In addition, increased [NADH] and [acetyl-CoA] also inhibit the oxidation of pyruvate to acetyl-CoA, and a high $[NADH] / [{NAD}^{+}]$ $left-bracket NADH right-bracket slash left-bracket NAD Superscript plus Baseline right-bracket$ ratio inhibits the dehydrogenase reactions of the citric acid cycle (see Fig. 16-18).

A yellow box labeled glucose has an arrow labeled blue highlighted hexokinase that points down to a yellow box labeled glucose 6-phosphate. A dashed arrow points from glucose 6-phosphate to a red circle containing a red “X” next to the arrow labeled blue highlighted hexokinase. A green circle containing a green triangle to the right of the same arrow is labeled P i. A short arrow point down from glucose 6-phosphate to a longer downward arrow labeled blue highlighted phosphofructokinase-1, which points to a yellow box labeled fructose 1,6-bisphosphate. Next to this arrow, a green circle containing a green triangle is next to A M P, A D P and a red circle containing a red “X” is next to A T P, citrate. An arrow labeled multistep points down to a yellow box labeled phosphoenolpyruvate, from which an arrow labeled blue highlighted pyruvate kinase points down to a yellow box labeled pyruvate. Next to the arrow labeled pyruvate kinase, a green circle containing a green triangle is next to A D P and a red circle containing a red “X” is next to A T P, N A D H. All of the reactions between glucose and pyruvate are enclosed in a bracket labeled glycolysis. From pyruvate, an arrow labeled blue highlighted pyruvate dehydrogenase complex points down to a yellow box labeled acetyl Co A. A dashed arrow points up from acetyl Co A to a red circle containing a red “X” next to this arrow. To the right of this arrow, a green circle containing a green triangle is next to A M P, A D P, N A D plus and a red circle containing a red “X” is next to A T P, N A D H. An arrow labeled blue highlighted citrate synthase points down from acetyl Co A to a yellow box labeled citrate. A dashed arrow points up from citrate to a red circle containing a red “X” next tot his arrow. To the right, a green circle containing a green triangle is next to A D P and a red circle containing a red “X” is next to A T P, N A D H. A short arrow points down from citrate, followed by a longer arrow labeled blue highlighted isocitrate dehydrogenase that points down to a yellow box labeled alpha-ketoglutarate. Next to this arrow, a green circle containing a green triangle is next to A D P and a red circle containing a red “X” is next to A T P. An arrow labeled alpha-ketoglutarate dehydrogenase points down to a yellow box labeled succinyl Co A, from which a dashed arrow points up to a red circle containing a red “X” next to the arrow labeled alpha-ketoglutarate dehydrogenase. Next to this arrow, there is a red circle containing a red “X” next to A T P, N A D H. An arrow labeled multistep points down from succinyl Co A to oxaloacetate. All of the reactions from acetyl Co A to oxaloacetate are enclosed in a bracket labeled citric acid cycle. A blue rectangle labeled respiratory chain has a green circle around a green triangle on top labeled A D P, P i. On its left side, a curved arrow shows the addition of an orange oval labeled N A D H and the loss of N A D plus. Below, a curved arrow shows the addition of A D P plus P i and the loss of an orange oval labeled A T P. To the right, a curved arrow shows the addition of one-half O 2 and loss of H 2 O. A bracket enclosing these reactions reads, oxidative phosphorylation.

Phosphofructokinase-1 is also inhibited by citrate, the first intermediate of the citric acid cycle. When the cycle is “idling,” citrate accumulates within mitochondria, then is transported into the cytosol. When the cytosolic concentrations of both ATP and citrate rise, they produce a concerted allosteric inhibition of phosphofructokinase-1 that is greater than the sum of their individual effects, slowing glycolysis.

SUMMARY 19.3 Regulation of Oxidative Phosphorylation

Oxidative phosphorylation is regulated by cellular energy demands. Intracellular [ADP] and the mass-action ratio $[ATP] / ([ADP] [P_{i}])$ $left-bracket ATP right-bracket slash left-parenthesis left-bracket ADP right-bracket left-bracket upper P Subscript i Baseline right-bracket right-parenthesis$ are measures of a cell’s energy status.
In hypoxic (oxygen-deprived) cells, a protein inhibitor blocks ATP hydrolysis by the reverse activity of ATP synthase, preventing a drastic drop in [ATP].
The adaptive responses to hypoxia, mediated by HIF-1, slow electron transfer into the respiratory chain and modify Complex IV to act more efficiently under low-oxygen conditions.
ATP and ADP concentrations set the rate of electron transfer through the respiratory chain via a series of coordinated controls on respiration, glycolysis, and the citric acid cycle.