Why is NADH equal to 3 ATP?

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Hint: 3 molecules of ATP generated by one molecule NADH in prokaryotes. In case of eukaryotes, 2 - 3 ATP molecules are generated per NADH molecule, for each electron pair is transferred to the electron transport chain.

Complete answer:
The theoretical maximum yield of ATP for the oxidation of one molecule of glucose during aerobic respiration is 38.
Determining the exact yield of ATP for aerobic respiration is difficult for a number of reasons. The number of ATP generated per NADH is not always a whole number. In eukaryotic cells, unlike prokaryotes, NADH generated in the cytoplasm during glycolysis must be transported across the mitochondrial membrane before it can transfer electrons to the electron transport chain which requires energy. As a result, 1-2 ATP are generated from these NADH.
However, considering the theoretical maximum yield of ATP per glucose molecule oxidized by aerobic respiration, for each pair of electrons transferred to the electron transport chain by one molecule of NADH, 3 ATP will be generated.
Total theoretical maximum number of ATP generated per Glucose in Prokaryotes = 38 ATP: 4 from substrate-level phosphorylation + 34 from oxidative phosphorylation.
The ATP yield by NADH is as follows –
Glycolysis : 2 NADH yields 6 ATP by oxidative phosphorylation
Transition Reaction : 2 NADH yields 6 ATP
Citric acid cycle : 6 NADH yields 18 ATP by oxidative phosphorylation
In eukaryotic cells, the theoretical maximum yield of ATP generated per glucose is 36 to 38, depending on how the 2 NADH generated in the cytoplasm during glycolysis enter the mitochondria and whether the resulting yield is 2 or 3 ATP per NADH.
Thus, from the above information, we can conclude two things:
In cytoplasm, one molecule of NADH is equivalent to 2 ATP.
Inside the mitochondria, one molecule of NADH is equivalent to 3 ATP.

Note:
NADH generated in the cytoplasm during glycolysis must be transported across the mitochondrial membrane before it can transfer electrons to the electron transport chain and this requires energy. As a result, between 1 and 2 ATP are generated from these NADH. Also ATP generated in eukaryotes & prokaryotes can be different.

Most of the usable energy obtained from the breakdown of carbohydrates or fats is derived by oxidative phosphorylation, which takes place within mitochondria. For example, the breakdown of glucose by glycolysis and the citric acid cycle yields a total of four molecules of ATP, ten molecules of NADH, and two molecules of FADH2 (see Chapter 2). Electrons from NADH and FADH2 are then transferred to molecular oxygen, coupled to the formation of an additional 32 to 34 ATP molecules by oxidative phosphorylation. Electron transport and oxidative phosphorylation are critical activities of protein complexes in the inner mitochondrial membrane, which ultimately serve as the major source of cellular energy.

The Electron Transport Chain

During oxidative phosphorylation, electrons derived from NADH and FADH2 combine with O2, and the energy released from these oxidation/ reduction reactions is used to drive the synthesis of ATP from ADP. The transfer of electrons from NADH to O2 is a very energy-yielding reaction, with ΔG°´ = -52.5 kcal/mol for each pair of electrons transferred. To be harvested in usable form, this energy must be produced gradually, by the passage of electrons through a series of carriers, which constitute the electron transport chain. These carriers are organized into four complexes in the inner mitochondrial membrane. A fifth protein complex then serves to couple the energy-yielding reactions of electron transport to ATP synthesis.

Electrons from NADH enter the electron transport chain in complex I, which consists of nearly 40 polypeptide chains (Figure 10.8). These electrons are initially transferred from NADH to flavin mononucleotide and then, through an iron-sulfur carrier, to coenzyme Q—an energy-yielding process with ΔG°´ = -16.6 kcal/mol. Coenzyme Q (also called ubiquinone) is a small, lipid-soluble molecule that carries electrons from complex I through the membrane to complex III, which consists of about ten polypeptides. In complex III, electrons are transferred from cytochrome b to cytochrome c—an energy-yielding reaction with ΔG°´ = -10.1 kcal/mol. Cytochrome c, a peripheral membrane protein bound to the outer face of the inner membrane, then carries electrons to complex IV (cytochrome oxidase), where they are finally transferred to O2 (ΔG°´ = -25.8 kcal/mol).

A distinct protein complex (complex II), which consists of four polypeptides, receives electrons from the citric acid cycle intermediate, succinate (Figure 10.9). These electrons are transferred to FADH2, rather than to NADH, and then to coenzyme Q. From coenzyme Q, electrons are transferred to complex III and then to complex IV as already described. In contrast to the transfer of electrons from NADH to coenzyme Q at complex I, the transfer of electrons from FADH2 to coenzyme Q is not associated with a significant decrease in free energy and, therefore, is not coupled to ATP synthesis. Consequently, the passage of electrons derived from FADH2 through the electron transport chain yields free energy only at complexes III and IV.

Figure 10.9

Transport of electrons from FADH2. Electrons from succinate enter the electron transport chain via FADH2 in complex II. They are then transferred to coenzyme Q and carried through the rest of the electron transport chain as described in Figure 10.8. The (more...)

The free energy derived from the passage of electrons through complexes I, III, and IV is harvested by being coupled to the synthesis of ATP. Importantly, the mechanism by which the energy derived from these electron transport reactions is coupled to ATP synthesis is fundamentally different from the synthesis of ATP during glycolysis or the citric acid cycle. In the latter cases, a high-energy phosphate is transferred directly to ADP from the other substrate of an energy-yielding reaction. For example, in the final reaction of glycolysis, the high-energy phosphate of phosphoenolpyruvate is transferred to ADP, yielding pyruvate plus ATP (see Figure 2.32). Such direct transfer of high-energy phosphate groups does not occur during electron transport. Instead, the energy derived from electron transport is coupled to the generation of a proton gradient across the inner mitochondrial membrane. The potential energy stored in this gradient is then harvested by a fifth protein complex, which couples the energetically favorable flow of protons back across the membrane to the synthesis of ATP.

Chemiosmotic Coupling

The mechanism of coupling electron transport to ATP generation, chemiosmotic coupling, is a striking example of the relationship between structure and function in cell biology. The hypothesis of chemiosmotic coupling was first proposed in 1961 by Peter Mitchell, who suggested that ATP is generated by the use of energy stored in the form of proton gradients across biological membranes, rather than by direct chemical transfer of high-energy groups. Biochemists were initially highly skeptical of this proposal, and the chemiosmotic hypothesis took more than a decade to win general acceptance in the scientific community. Overwhelming evidence eventually accumulated in its favor, however, and chemiosmotic coupling is now recognized as a general mechanism of ATP generation, operating not only in mitochondria but also in chloroplasts and in bacteria, where ATP is generated via a proton gradient across the plasma membrane.

Electron transport through complexes I, III, and IV is coupled to the transport of protons out of the interior of the mitochondrion (see Figure 10.8). Thus, the energy-yielding reactions of electron transport are coupled to the transfer of protons from the matrix to the intermembrane space, which establishes a proton gradient across the inner membrane. Complexes I and IV appear to act as proton pumps that transfer protons across the membrane as a result of conformational changes induced by electron transport. In complex III, protons are carried across the membrane by coenzyme Q, which accepts protons from the matrix at complexes I or II and releases them into the intermembrane space at complex III. Complexes I and III each transfer four protons across the membrane per pair of electrons. In complex IV, two protons per pair of electrons are pumped across the membrane and another two protons per pair of electrons are combined with O2 to form H2O within the matrix. Thus, the equivalent of four protons per pair of electrons are transported out of the mitochondrial matrix at each of these three complexes. This transfer of protons from the matrix to the intermembrane space plays the critical role of converting the energy derived from the oxidation/reduction reactions of electron transport to the potential energy stored in a proton gradient.

Because protons are electrically charged particles, the potential energy stored in the proton gradient is electric as well as chemical in nature. The electric component corresponds to the voltage difference across the inner mitochondrial membrane, with the matrix of the mitochondrion negative and the intermembrane space positive. The corresponding free energy is given by the equation

where F is the Faraday constant and ΔV is the membrane potential. The additional free energy corresponding to the difference in proton concentration across the membrane is given by the equation

where [H+]i and [H+]o refer, respectively, to the proton concentrations inside and outside the mitochondria.

In metabolically active cells, protons are typically pumped out of the matrix such that the proton gradient across the inner membrane corresponds to about one pH unit, or a tenfold lower concentration of protons within mitochondria (Figure 10.10). The pH of the mitochondrial matrix is therefore about 8, compared to the neutral pH (approximately 7) of the cytosol and intermembrane space. This gradient also generates an electric potential of approximately 0.14 V across the membrane, with the matrix negative. Both the pH gradient and the electric potential drive protons back into the matrix from the cytosol, so they combine to form an electrochemical gradient across the inner mitochondrial membrane, corresponding to a ΔG of approximately -5 kcal/mol per proton.

Figure 10.10

The electrochemical nature of the proton gradient. Since protons are positively charged, the proton gradient established across the inner mitochondrial membrane has both chemical and electric components. The chemical component is the proton concentration, (more...)

Because the phospholipid bilayer is impermeable to ions, protons are able to cross the membrane only through a protein channel. This restriction allows the energy in the electrochemical gradient to be harnessed and converted to ATP as a result of the action of the fifth complex involved in oxidative phosphorylation, complex V, or ATP synthase (see Figure 10.8). ATP synthase is organized into two structurally distinct components, F0 and F1, which are linked by a slender stalk (Figure 10.11). The F0 portion spans the inner membrane and provides a channel through which protons are able to flow back from the intermembrane space to the matrix. The energetically favorable return of protons to the matrix is coupled to ATP synthesis by the F1 subunit, which catalyzes the synthesis of ATP from ADP and phosphate ions (Pi). Detailed structural studies have established the mechanism of ATP synthase action, which involves mechanical coupling between the F0 and F1 subunits. In particular, the flow of protons through F0 drives the rotation of F1, which acts as a rotary motor to drive ATP synthesis.

Figure 10.11

Structure of ATP synthase. The mitochondrial ATP synthase (complex V) consists of two multisubunit components, F0 and F1, which are linked by a slender stalk. F0 spans the lipid bilayer, forming a channel through which protons can cross the membrane. (more...)

It appears that the flow of four protons back across the membrane through F0 is required to drive the synthesis of one molecule of ATP by F1, consistent with the proton transfers at complexes I, III, and IV each contributing sufficient free energy to the proton gradient to drive the synthesis of one ATP molecule. The oxidation of one molecule of NADH thus leads to the synthesis of three molecules of ATP, whereas the oxidation of FADH2, which enters the electron transport chain at complex II, yields only two ATP molecules.

Transport of Metabolites across the Inner Membrane

In addition to driving the synthesis of ATP, the potential energy stored in the electrochemical gradient drives the transport of small molecules into and out of mitochondria. For example, the ATP synthesized within mitochondria has to be exported to the cytosol, while ADP and Pi need to be imported from the cytosol for ATP synthesis to continue. The electrochemical gradient generated by proton pumping provides energy required for the transport of these molecules and other metabolites that need to be concentrated within mitochondria (Figure 10.12).

Figure 10.12

Transport of metabolites across the mitochondrial inner membrane. The transport of small molecules across the inner membrane is mediated by membrane-spanning transport proteins and driven by the electrochemical gradient. For example, ATP is exported from (more...)

The transport of ATP and ADP across the inner membrane is mediated by an integral membrane protein, the adenine nucleotide translocator, which transports one molecule of ADP into the mitochondrion in exchange for one molecule of ATP transferred from the mitochondrion to the cytosol. Because ATP carries more negative charge than ADP (-4 compared to -3), this exchange is driven by the voltage component of the electrochemical gradient. Since the proton gradient establishes a positive charge on the cytosolic side of the membrane, the export of ATP in exchange for ADP is energetically favorable.

The synthesis of ATP within the mitochondrion requires phosphate ions (Pi) as well as ADP, so Pi must also be imported from the cytosol. This is mediated by another membrane transport protein, which imports phosphate (H2PO4-) and exports hydroxyl ions (OH-). This exchange is electrically neutral because both phosphate and hydroxyl ions have a charge of -1. However, the exchange is driven by the proton concentration gradient; the higher pH within mitochondria corresponds to a higher concentration of hydroxyl ions, favoring their translocation to the cytosolic side of the membrane.

Energy from the electrochemical gradient is similarly used to drive the transport of other metabolites into mitochondria. For example, the import of pyruvate from the cytosol (where it is produced by glycolysis) is mediated by a transporter that exchanges pyruvate for hydroxyl ions. Other intermediates of the citric acid cycle are able to shuttle between mitochondria and the cytosol by similar exchange mechanisms.

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Key Experiment: The Chemiosmotic Theory.

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