The Respiratory Chain: Electrons, Protons, and ATP Production

Pyruvate oxidation and the operation of the citric acid cycle generate large amounts of reduced electron carriers containing trapped energy. To liberate this energy and produce ATP, something must happen to these reduced carriers. Furthermore, without NAD+ and FAD, the oxidative steps of glycolysis, pyruvate oxidation, and the citric acid cycle could not occur. To regenerate NAD+ and FAD, the reduced forms of these carriers must have some way to get rid of their hydrogens (H+ + e–). The fate of these protons and electrons is the rest of the story of cellular respiration. The story hasthree parts:

1. The electrons pass through a series of membrane-associated electron carriers called the respiratory chain or the electron transport chain.

2. The flow of electrons along the chain accomplishes the active transport of protons across the inner mitochondrial membrane, out of the matrix, creating a proton concentration gradient.

3. The protons diffuse back into the mitochondrial matrix through a proton channel, which couples this diffusion to the synthesis of ATP.

The overall process of ATP synthesis resulting from electron transport through the chain is called oxidative phosphorylation.

Before we proceed with the details of oxidative phosphorylation, let’s reflect on an important question: Why should the respiratory chain have so many components and complex processes? Why, for example, don’t cells use the following single step?

NADH + H+ + 1⁄2 O2 ®NAD+ + H2O

Fundamentally, this would be an untamable reaction. It would be very exergonic—rather like setting off a stick of dynamite in the cell. There is no biochemical way to harvest that burst of energy efficiently and put it to physiological use (that is, no metabolic reaction that is so endergonic as to consume a significant fraction of that energy in a single step). To control the release of energy during the oxidation of glucose in a cell, evolution has produced the lengthy electron transport chain we observe today: a series of reactions, each releasing a small, manageable amount of energy.

The respiratory chain transports electrons and releases energy

The respiratory chain contains large integral proteins, smaller mobile proteins, and even a smaller lipid molecule:

Four large protein complexes containing carrier molecules and their associated enzymes are integral proteins of the inner mitochondrial membrane in eukaryotes.

Cytochrome c is a small peripheral protein that lies in the space between the inner and outer mitochondrial membranes. It is loosely attached to the inner membrane.

A nonprotein component called ubiquinone(abbreviated Q) is a small, nonpolar molecule that moves freely within the hydrophobic interior of the phospholipids bilayer of the inner membrane.

NADH + H+ passes electrons to Q by way of the firstlarge protein complex, called NADH-Q reductase, which contains twenty-six polypeptides and attached prosthetic groups. NADH-Q reductase passes the electrons to Q, forming QH₂. The second complex, succinate dehydrogenase, passes electrons to Q from FADH₂ during the formation of fumarate from succinate in reaction 6 of the citric acid cycle. These electrons enter the chain later than those from NADH. The third complex, cytochrome c reductase, with ten subunits, receives electrons from QH₂ and passes them to cytochrome c. The fourth complex, cytochrome c oxidase, with eight subunits, receives electrons from cytochrome c and passes them to oxygen, which with these extra electrons (1⁄2 O₂ –) picks up two hydrogen ions (H+) to form H₂O. The electron carriers of the respiratory chain (including those contained in the three protein complexes) differ as to how they change when they become reduced. NAD+, for example, accepts H– (a hydride ion — one proton and two electrons), leaving the proton from the other hydrogen atom to float free: NADH + H+. Other carriers, including Q, bind both protons and both electrons becoming, for example, QH2. The remainder of the chain, however, is only an electron transport process. Electrons, but not protons, are passed from Q to cytochrome c. An electron from QH₂ reduces a cyto-chrome’s Fe3+ to Fe2+. The fate of the protons will be discussed below. Electron transport within each of the three protein complexes results, as we’ll see, in the pumping of protons across the inner mitochondrial membrane, and the return of the protons across the membrane is coupled to the formation of ATP. Thus the energy originally contained in glucose and other foods is finally captured in the cellular energy currency, ATP. For each pair of electrons passed along the chain from NADH + H+ to oxygen, three molecules of ATP are formed. If only electrons are carried through the final reactions of the respiratory chain, what happens to the protons? How are proton movements coupled to the production of ATP?

Proton diffusion is coupled to ATP synthesis

As we have seen, all the carriers and enzymes of the respiratory chain except cytochrome c are embedded in the inner mitochondrial membrane. The operation of the respiratory chain results in the active transport of protons (H+), against their concentration gradient, across the inner membrane of the mitochondrion from the mitochondrial matrix to the intermembrane space (the space between the inner and outer mitochondrial membranes). This occurs because the electron carriers contained in the three large protein complexes are arranged such that protons are taken up on one side of the membrane (the mitochondrial matrix) and transported along with electrons to the other side (the intermembrane space). Thus, the protein complexes act as proton pumps. Because of the positive charge on the protons (H+), this transport causes not only a difference in proton concentration, but also a difference in electric charge, across the membrane, with the inside of the organelle (the matrix) more negative than the intermembrane space.

Together, the proton concentration gradient and the charge difference constitute a source of potential energy called the proton-motive force. This force tends to drive the protons back across the membrane, just as the charge on a battery drives the flow of electrons discharging the battery. The conversion of the proton-motive force into kinetic energy is prevented by the fact that protons cannot cross the hydrophobic lipid bilayer of the inner membrane by simple diffusion.

However, they can diffuse across the membrane by passing through a specific proton channel, called ATP synthase, that couples proton movement to the synthesis of ATP. This coupling of proton-motive force and ATP synthesis is called the chemiosmotic mechanism, or chemiosmosis.

The chemiosmotic mechanism for ATP synthesis

The chemiosmotic mechanism uses ATP synthase to couple proton diffusion to ATP synthesis. This mechanism has three parts:

1. The flow of electrons from one electron carrier to another in the respiratory chain is a series of exergonic reactions that occurs in the inner mitochondrial membrane.

2. These exergonic reactions drive the endergonic pumping of H+ out of the mitochondrial matrix and across the inner membrane into the intermembrane space. This pumping establishes and maintains a H+ gradient.

3. The potential energy of the H+ gradient or protonmotive force, is harnessed by ATP synthase. This protein has two roles: It acts as a channel allowing the H+ to diffuse back into the matrix and it uses the energy of that diffusion to make ATP from ADP and Pi. ATP synthesis is a reversible reaction and ATP synthase can also act as an ATPase, hydrolyzing ATP to ADP and Pi: ATP®ADP + Pi + free energy

If the reaction goes to the right, free energy is released, and that energy is used to pump H+ out of the mitochondrial matrix. If the reaction goes to the left, it uses free energy from H+ diffusion into the matrix to make ATP. What makes it prefer ATP synthesis? There are two answers to this question.

ATP leaves the mitochondrial matrix for use elsewhere in the cell as soon as it is made, keeping the ATP concentration in the matrix low and driving the reaction toward the left. A person hydrolyzes about 1025 ATP molecules per day, and clearly the vast majority are recycled using the free energy from the oxidation of glucose.

The H+ gradient is maintained by electron transport and proton pumping. (The electrons, you will recall, come from the oxidation of NADH and FADH₂, which are themselves reduced by the oxidations of glycolysis and the citric acid cycle. Thus, one reason you eat is to replenish the H+ gradient!) ATP synthase is a large multi-protein machine, containing 16 different polypeptides in mammals. It has two functional components. One of these components is the membrane channel for H+. The other component sticks out into the mitochondrial matrix like a lollipop and contains the active site for ATP synthesis. The actual mechanism of transferring energy from the H+ gradient to the phosphorylation of ADP involves the physical rotation of the core of the enzyme, with this rotational energy transferred to ATP.

Experiments demonstrate chemiosmosis

Two key experiments demonstrated (1) that a proton (H+) gradi ent across a membrane can drive ATP synthesis; and (2) that the enzyme ATP synthase is the catalyst for this reaction.

Experiment 1 tested the hypothesis that ATP synthesis is driven by the H+ gradient across an inner mitochondrial membrane. In this experiment, mitochondria without a food source were “fooled” into making ATP when researchers raised the H+ concentration in their environment. A sample of isolated mitochondria that had been exposed to a low H+ concentration was suddenly put in a medium with a high concentration of H+. The outer mitochondrial membrane, unlike the inner one, is freely permeable to H+, so H+ rapidly diffused into the intermembrane space. This created an artificial gradient across the inner membrane, which the mitochondria used to make ATP from ADP and Pi. This result supported the hypothesis and provided strong evidence for chemiosmosis.

Experiment 2 tested the hypothesis that the enzyme ATPase couples a proton gradient to ATP synthesis. In this experiment, a proton pump isolated from a bacterium was added to artificial membrane vesicles. When an appropriate energy source was provided, H+ was pumped into the vesicles, creating a gradient. If mammalian ATP synthase was then inserted into the membranes of these vesicles and the energy source removed, the vesicles made ATP even in the absence of the usual electron carriers. Again, the result supported the hypothesis, showing that the enzyme ATP synthase is the coupling factor in the membrane.