Introduction and Goals
This tutorial will describe the mechanisms involved in the synthesis of ATP during cellular respiration. The final stage of cellular respiration occurs in the mitochondria, where the reduced electron carriers (NADH and FADH2) donate their electrons to oxygen via an electron transport chain. As the electrons travel, a hydrogen electrochemical gradient is generated across one of the two mitochondrial membranes. The energy of this electrochemical proton gradient is used to synthesize ATP.
By the end of this tutorial you should know:
- The nature of the chemiosmotic coupling of electron transport and ATP synthesis
- The features of a mitochondrion
- The meaning of redox potential and how it determines the flow of electrons
- The mechanisms of electron transport (including the role of the reduced electron carriers NADH and FADH2, and oxygen)
- How the hydrogen electrochemical gradient is formed
- How the hydrogen electrochemical gradient is used to synthesize ATP
The chemiosmotic coupling of electron transport and ATP synthesis
The majority of cellular ATP is synthesized during cellular respiration in the mitochondria of animal and plant cells, and during photosynthesis in the chloroplasts of plant and algal cells. The mechanism of ATP synthesis in these two organelles is very similar and represents an evolutionarily conserved mechanism (which bacteria also employ). Basically this mechanism is two linked processes: electron transport and ATP synthesis (both are mediated by membrane-bound proteins). Electrons are transferred along an electron transport chain (ETC) that is composed of protein complexes embedded in a membrane. As the electrons move from one protein to another, protons are pumped across the membrane. This results in an electrochemical proton gradient, where the free energy released during electron movement is captured in this gradient. The hydrogen ions then flow back across the membrane (down the electrochemical gradient) through a specialized protein complex termed the ATP synthase, which captures the free energy of the hydrogen gradient to drive the synthesis of ATP from ADP and inorganic phosphate. The linkage of electron transport, proton pumping and ATP synthesis is referred to as chemiosmotic coupling. In mitochondria this process is the final stage of cellular respiration and is referred to as oxidative phosphorylation. In chloroplasts this process is often referred to as the light reactions of photosynthesis. This tutorial will describe oxidative phosphorylation in detail. Photosynthesis will be described in the next tutorial.
Oxidative phosphorylation occurs in the mitochondria
The complete oxidation of glucose into CO2occurs during glycolysis and the citric acid cycle (both of which were described in the previous tutorial); you may recall that very little energy, in the form of ATP, is actually produced in these two pathways (see Fig. 5; in Glycolysis, Fermentation and the Citric Acid Cycle tutorial). The majority of energy, at this point, is stored in the reduced electron carriers NADH and FADH2. They will be used in the final stage of cellular respiration, namely oxidative phosphorylation, which occurs in mitochondria. A mitochondrion is an organelle surrounded by two membranes (an inner membrane and an outer membrane; Figure 1). These two membranes define the intermembrane space, and the matrix (the very interior space of the mitochondrion) is defined by the inner membrane. The outer membrane is very permeable, and it allows many molecules to flow in and out of the mitochondrion. The inner membrane, which is much less permeable, is the site of oxidative phosphorylation. The proteins of the ETC and the ATP synthase are embedded in the inner membrane, which has numerous distinct infoldings (termed cristae). Cristae increase the inner membrane's surface area. The matrix is the site of the citric acid cycle. Mitochondria also contain their own genomes and can transcribe mRNAs and translate proteins.
In oxidative phosphorylation, the reduced electron carriers (NADH and FADH2) donate their electrons to the proteins of the ETC. The electrons are passed from protein to protein in a stepwise fashion, coupled to proton pumping across the inner mitochondrial membrane, into the intermembrane space. Eventually the electrons are donated to oxygen to form water, hence the necessity for oxygen in cellular respiration. The direction of the flow of electrons along the ETC is determined by the affinity of different carriers for electrons. This affinity can be expressed as a redox potential, an empirical measure of the affinity of any pair of oxidized and reduced compounds (redox pair) for electrons. For example, the reaction NAD+ + 2H+ + 2e- -> NADH (NADH and NAD+ are the redox pair) has a redox potential of -320mV; this negative value indicates that NADH is a good electron donor (it has a relatively low affinity for electrons). In contrast, the reaction 1/2 O2 + 2H+ + 2e- -> H2O has a redox potential of +820mV, indicating that oxygen is an excellent electron acceptor (it has a high affinity for electrons). In the ETC, the direction of electron flow is from compounds with lower redox potentials to compounds with successively higher redox potentials; that is, starting with NADH or FADH2 and terminating with oxygen (see Figure 2). When electrons are passed from an electron donor (e.g. NADH) to an electron acceptor (e.g. O2, the free energy released can be calculated from the difference between the redox potential of one redox pair (e.g. NADH and NAD+) and the other redox pair.
The Respiratory Chain
The mitochondrial electron transport chain, also referred to as the respiratory chain, is organized into three multiprotein complexes: NADH dehydrogenase, cytochrome b-c complex, and cytochrome oxidase. Each respiratory chain complex is composed of several different proteins capable of both electron transport and the pumping of protons across the inner membrane, thereby generating an electrochemical proton gradient (illustrated in Figure 3). The proteins within these complexes have a variety of prosthetic groups (see tutorial Properties of Macromolecules 1-Proteins), including iron-sulfur centers, hemes, flavins (the multiple-ringed moieties of FADs) and copper, all of which are capable of accepting and donating electrons. Electron transport is initiated when a pair of electrons and a proton are released from NADH and accepted by NADH dehydrogenase, whereupon the electrons are transported from one protein to another within the complex. The electrons are then transported from that complex to the cytochrome b-c complex by the mobile electron carrier coenzyme Q (CoQ), also called ubiquinone, the sole non-protein electron carrier in the respiratory chain. The electrons move through the cytochrome b-c complex and are transported to the final complex by the small protein cytochrome c. The final complex, cytochrome oxidase, catalyzes the transfer of the electrons to oxygen. The free energy of this complete reaction (NADH + H+ + 1/2 O2 -> NAD+ + H2O) is -52.6 kcal/mol. The free energy is released in a stepwise fashion as the electrons move through the ETC, and is captured in the electrochemical proton gradient. FADH2also donates its electrons to the respiratory chain, but because its redox potential is higher than that of NADH dehydrogenase, it cannot donate its electrons to that protein complex. Instead, the electrons from FADH2 are donated to succinate dehydrogenase, which, in turn, will pass the electrons to CoQ and they will be transported through the remainder of the respiratory chain. The hydrogen electrochemical gradient that is generated during electron transport initiated by FADH2 is not as great as that generated by NADH. This is because FADH2donates its electrons to succinate dehydrogenase, which does not pump any protons, and the electrons bypass NADH dehydrogenase, which does pump protons.
The Electrochemical Proton Gradient
Coordinately with electron transport, the three respiratory protein complexes described above pump protons from the matrix into the intermembrane space, resulting in an electrochemical gradient across the inner membrane. As electrons move through these multi-protein complexes, they are frequently paired with a proton (H+) to neutralize their charge as they move from one side of the membrane to the other. Allosteric changes in the protein complexes can also result in the pumping of protons across the membrane. The pumping of protons generates a gradient across the inner membrane of one pH unit difference (a ten-fold difference in hydrogen ion concentration) between the matrix and the intermembrane space. In addition, a membrane potential is generated due to a positive charge in the intermembrane space and the net negative charge in the matrix. The electrochemical gradient across the inner membrane is comprised of both the pH gradient and membrane potential and is the force, often referred to as the proton motive force (PMF), which will drive protons back across the inner membrane into the matrix and, in doing so, drive ATP synthesis.
The PMF is used to drive ATP synthesis via the ATP synthase, a protein complex that converts the energy of the proton gradient into chemical bonds. The ATP synthase has two distinct subunits: the transmembrane F0 subunit, which contains a protein channel for the flow of protons; and the F1 subunit, which protrudes into the matrix space and catalyzes the synthesis of ATP from ADP and inorganic phosphate (Figure 4). A portion of the F1 subunit termed the stalk links the two subunits. As protons flow through the channel in the F0 subunit, they cause the embedded stalk to rotate in the stationary F1 subunit, thereby converting the energy of the electrochemical gradient into mechanical energy. As the stalk rotates in one direction, it induces conformational changes in the proteins of the F1 subunit, which, in turn, catalyze the synthesis of ATP - thereby converting the mechanical energy of stalk rotation to chemical bond energy. Approximately three protons must pass through the ATP synthase complex for one ATP molecule to be synthesized.
The Proton Gradient
The proton gradient as the link between electron transport and ATP synthesis
The proton gradient is critical to the chemiosmotic coupling of electron transport and ATP synthesis. As electrons are moved along the respiratory chain, protons are pumped across the inner membrane, from the matrix into the intermembrane space; this results in an electrochemical proton gradient. The force of this gradient drives protons back across the inner membrane into the matrix, through the F0 subunit of the ATP synthase, which results in activation of the F1 subunits and synthesis of ATP. On average, for each NADH, approximately 3 ATPs are synthesized, and for each FADH2, approximately 2 ATPs are synthesized. The lower ATP yield for FADH2 results from the smaller proton gradient generated when electrons are donated from FADH2. In this case, the electrons are donated to succinate dehydrogenase, which does not pump protons when electrons enter the ETC, thereby bypassing NADH dehydrogenase and the protons it would pump across the membrane.
Oxidative phosphorylation is a mechanism for ATP synthesis in both plant and animal cells. It involves the chemiosmotic coupling of electron transport and ATP synthesis. Oxidative phosphorylation occurs in the mitochondria. The mitochondrion has two membranes: an inner membrane and an outer membrane. The space defined by the inner membrane is the matrix, and the space between the two membranes is the intermembrane space. NADH and FADH2, generated in glycolysis and the citric acid cycle, are oxidized in the mitochondria. They donate their electrons to protein complexes embedded in the mitochondrial inner membrane, composed of many polypeptides with a variety of prosthetic groups capable of accepting and donating electrons. These complexes are components of the respiratory chain. Electrons donated from NADH and FADH2are transported along the respiratory chain and they will eventually be donated to oxygen, thus generating water. The direction of electron transport is determined by the redox potential of each potential electron carrier. There are three major protein complexes that participate in the respiratory chain that transports electrons and that pump hydrogen ions across the inner membrane, hence resulting in a hydrogen electrochemical gradient. This electrochemical gradient generates a proton motive force (PMF) that drives the hydrogen ions back across the inner membrane through the ATP synthase. The ATP synthase is composed of two subunts: the F0 subunit, which provides a channel for the flow of hydrogen ions back across the inner membrane; and the F1 subunit, which catalyzes the synthesis of ATP from ADP + Pi. As hydrogen ions flow through the F0 subunit, a portion of the subunit rotates in the membrane. As it rotates, it induces conformational changes in the F1 subunit that activate the ATP synthesis activity, thereby converting the free energy of the hydrogen electrochemical gradient (generated by the proteins of the electron transport chain) into the energy of a chemical bond.