Oxidation of glucose may be broken into two phases (See Figure 15.1):
1. Oxidation of the glucose carbon atoms
2. Reduction of molecular oxygen
Overview: The electron transport system (ETS) is a mechanism for capturing in the form of ATP, energy from the transfer of electrons from NADH and FADH2 to O2. During this process, the mitochondrion expels protons, creating a pH gradient across the mitochondrial membrane. When the proton gradient energy is harnessed (oxidative phosphorylation), ATP is produced from ADP and Pi.
Electrons are carried to the electron transport chain by the molecules NADH and FADH2, which are produced in many cellular processes, included glycolysis and citric acid cycle (See Figure 15.2b). Reoxidation of NADH (release of electrons) yields 3 ATPs. Reoxidation of FADH2 yields 2 ATPs. Thus, oxidation of one molecule of glucose yields 38 ATPs from glycolysis and the citric acid cycle.
Electron transport and oxidative phosphorylation occur in the mitochondrion, the cell's "power plant". The citric acid enzymes are housed there as is pyruvate dehydrogenase, enzymes for fatty acid oxidation, and all of the enzymes and proteins of the electron transport/oxidative phosphorylation system.
Mitochondria have a smooth outer membrane and a complicated inner membrane, with many folds inside the organelle (See Figure 15.2a). This infolding material is called cristae, the number of which vary with the respiratory needs of the cell. Cells with low respiration rates have fewer cristae; those with higher rates have more. Proteins of electron transport and oxidative phosphorylation are bound to the inner mitochondrial membrane. The "matrix" of the inner mitochondrial compartment (a gel-like aqueous material) contains high concentrations of oxidative metabolic enzymes (citric acid cycle, etc.) as well as the mitochondrial DNA, RNA, and ribosomes for making mitochondrial proteins.
The outer membrane of the mitochondrion contains porin, a pore-forming protein that allows diffusion of up to 10 kD (10,000 Dalton molecular weight) molecules. The inner membrane is much richer in proteins (up to 75% protein by mass) than the outer membrane and allows only O2, CO2 and H20 to freely pass.The inner membrane is the anchor for electron transport and oxidative phosphorylation proteins, and also contains transport proteins for passing common metabolites, such as ATP, ADP, pyruvate, Ca++, and phosphate. The integrity of the inner membrane is important for operation of the mitochondrion, which harnesses energy from the chemical gradients it creates across the membrane.
Electron transport involves transfer of the reducing equivalents of electrons through several biological compounds. As a compound accepts electrons, it is reduced, and as it passes the electron on to the next molecule in the chain the molecule is oxidized.
This may be written as a reaction:
Reduced molecule #1 + Oxidized Molecule #2 Oxidized Molecule #1 + Reduced Molecule #2
One example of this shown in your book is:
Fe(+2) + Cu (+2) Fe(+3) + Cu(+1)
The tendency of a molecule to act as an electron donor is referred to as the Reduction Potential, and is given the symbol 'E'. The Standard Reduction Potential at pH 7.0 is given the symbol . The scale for is set so that the potential of a hydrogen electrode is 0.00 volts. Table 15.1 shows the Standard Reduction Potential of many biological compounds. The Standard Reduction Potential voltages shown in the table can be viewed as the tendency of electrons to flow towards the molecule of interest. Molecules with a more positive Standard Reduction Potential will pull electrons away from molecules with a more negative Standard Reduction Potential.
As an example,
Fe(+3) + Cytochrome a3 (+2) Fe(+2) + Cytochrome a3 (+3)
The Standard Reduction Potentials shown in Table 15.1 predict that this reaction will go to the right if the concentration of all the molecules is the same. Students should note a few things here. First of all, a reaction such as this is called a Reduction/Oxidation reaction (or Redox reaction for short). Each side of the equation has a reductant and an oxidant. The reaction is an equilibrium, and is governed by the Gibbs free energy rules. Thus, under some conditions, the reaction will move to the left, and under other conditions it will move to the right. can be related to by the following equation:
where 'n' is the number of electrons transferred, F is Faraday's constant (96.5 kJ/mol/Volt), and = (acceptor molecule) - (donor molecule)
Rearranging into the G equation you've seen previously,
G = -nF + RTln (Products/Reactants)
It will can be noted from Figure 15.7 that the flow of electrons through the cell's electron transport chain is strictly in the direction of molecules with increasing Standard Reduction Potentials. Transfer of electrons in this manner releases energy that can be used to do the work of the cell.
The free energy released during the entire electron transfer process is sufficient in theory to yield 7 ATPs for each NADH. In reality, cells appear to synthesize about 3ATPs for each NADH, an efficiency of 42%. (Note: FADH2, another electron carrier, enters the electron transport system at a point different from NADH, and yields only 2 ATPs for each FADH2.) The transfer of electrons from NADH to O2 does not occur directly, but rather by a series of small steps three of which yield ATP. We call the process of making ATP by the capture of energy produced during electron transport Oxidative Phosphorylation.
In electron transport, electrons from NADH and FADH2 are passed through four complexes (I, II, III, IV) (See Figure 15.10). Complex V depicted in the figure refers to the process of oxidative phosphorylation. The sequence of events in ETS is as follows.
The sequence of passage of electrons through the ETS can be studied using chemicals that inhibit the ETS at specific points (See Figure 15.9). Rotenone inhibits ETS in complex I. The ETS can be restarted in the presence of rotenone if an FADH2 generating system is added (this bypasses the block in complex I). Antimycin blocks complex III (passage of electrons past cytochrome b). TMPD can be used to transfer electrons directly from ascorbate to cytochrome c. If this is employed, the Antimycin A block can be bypassed. Addition of cyanide completely inhibits the electron transport system by blocking transfer of electrons from cyt a3 to oxygen.
Energy produced by electrons entering the ETS from NADH, FADH2, or ascorbate result in synthesis respectively of 3,2, and 1 ATP per molecule. An oxygen electrode (an electrode capable of measuring oxygen concentrations in solutions) reveals a tight relationship between oxidative phosphorylation and electron transport. When mitochondria are placed in solution with no ETS inhibitors present, but also no ADP, the oxygen electrode registers a steady concentration of oxygen near mitochondria,indicating oxygen is not being used by the mitochondria. This is due to the fact that when there is no way for the mitochondria to make ATP, the ETS is blocked. Electron transport is thus coupled to oxidative phosphorylation. When ADP is added to the mitochondria in sufficient quantities, ETS begins, as measured by disappearance of oxygen from the solution, and the amount of oxygen used is consistent with the amount of ATP produced - 3 ATP per electrons entering at NADH, 2ATP per electrons entering at FADH2, and 1 ATP per electrons entering from ascorbate. There is a bit of controversy about the exact numbers of ATPs generated per NADH and FADH2. While we will note that the number needn't be exactly 3.0 and 2.0 per NADH and FADH2, we will use those numbers as correct.
The nicotinamides, flavins, and cytochromes of the ETS system absorb ultraviolet and visible light in distinctive ways. For each molecule, the absorption spectrum of the oxidized form of the molecule is different from the absorption spectrum of the reduced form of the molecule. If one takes the spectra of mitochondria in the fully oxidized or reduced form, and then subtracts one from the other, a "difference spectrum" is obtained (See Figure 15.8). By examining difference spectra of pure forms of each of the oxidized and reduced forms of the molecules, and comparing it to difference spectra of mitochondria in the fully oxidized or reduced form, it is possible to ascertain in mitochondria the percentage of each of the ETS molecules in the oxidized or reduced states.
Inner mitochondrial transport systems move the following molecules across the inner membrane:
Oxidation of pyruvate produced by glucose in glycolysis is accomplished by citric acid enzymes in the mitochondrion. This yields NADH, whose electrons can enter the electron transport system directly. NADH from glycolysis, however is in the cytosol. Since the mitochondrion does not have a direct transfer mechanism for NADH, "shuttles" are used instead, which ferry the electrons in (See Figure 15.11). Glycerophosphate (insect muscle) uses an enzyme in the outer layer of the inner mitochondrial membrane - Flavoprotein dehydrogenase (FD). In this series of reactions, dihydroxyacetone phosphate (DHAP) is reduced to 3-phosphoglycerol by 3-phosphoglycerol dehydrogenase as NADH is oxidixed to NAD+ (The electrons from NADH are transferred to 3-phosphoglycerol). In the second step, the electrons of 3-phosphoglycerol are transferred to FAD (Yielding FADH2) and DHAP is produced. The enzyme, FD is embedded in the outer mitochondrial membrane, and the electrons in FADH2 are transferred directly to the electron transport chain. This process, because it goes through FADH2, yields 2 ATP for each original NADH.
Another shuttle (mammals) is the malate-aspartate shuttle. This system relies on transport systems for malate, aspartate, glutamate, and alpha-keto-glutarate. Electrons from NADH outside the matrix are transferred in as follows (See Figure 15.11):
The complexes of the ETS are embedded in the inner mitochondrial membrane, where they have considerable ability to move independently of each other - they are not attached in any way.
Complex I passes electrons from NADH to Coenzyme Q (CoQ). The complex contains one molecule of flavin mononucleotide (FMN) - similar to FAD - and several iron-sulfur proteins (nonheme iron proteins). The sulfurs in these proteins come from Cys residues and from free sulfide. The iron atoms are surrounded each by 4 sulfur atoms. (See Figure 15.4) Remember that iron can exist as Fe++ and Fe+++. The difference is a single electron. Despite the presence of as many as four Fe atoms, the total charge difference between the oxidized and reduced forms of the cluster is one electron. For example, each four FeS cluster in ferredoxin has one Fe++ and three Fe+++ (total charge = +11) when oxidized, and two Fe++ and two Fe+++ (total charge = +10) when reduced.
FMN and CoQ can have three oxidation states (See Figure on page 529). NADH passes pairs of electrons. Both FMN and CoQ can accept and donate either one or two electrons. FMN and CoQ buffer the flow of electrons downstream because Complex III can only handle electrons one at a time. The tail of CoQ is hydrophobic, and is soluble in the inner mitochondrial membrane.
Complex II contains succinate dehydrogenase (citric acid enzyme), which passes electrons from succinate (through FADH2) to CoQ. Other components of the system include a four Fe-S cluster, two 2 Fe-S clusters, and a cytochrome b560. No ATP is generated in passing from complex II to CoQ.
The complex contains two cytochrome b's, a cytochrome c, and a single 2Fe-S cluster. The complex is scattered across the inner mitochondrial membrane. Cytochrome c1 and the Fe-S protein are on the membrane's outer surface, while cytochrome b is transmembranous. The cytochromes hold Fe with N instead of S, and the different cytochromes here have different configurations of the hemes (See Figure 15.6). The rings containing the N are called porphyrins, and the ring of cytochrome b (contains two) is called protoporphyrin IX, which is also used in hemoglobin.
Cytochromes are ubiquitous among aerobic organisms. The proteins contain heme groups that alternate between Fe++ and Fe+++ as above.
Cyt C is a peripheral membrane protein (different from cyt c1 above). It is only loosely bound to the outer surface of the inner membrane. It alternately binds cyt c1 of complex III and cyt c oxidase in Complex IV (passing electrons between them). The heme group of cyt c is buried in the protein. Lysine residues appear to mediate the binding to both cyt c1 and to cyt c oxidase.
Cyt c Oxidase (Complex IV) catalyzes one electron oxidations of four consecutive reduced cytochrome c molecules and the four electron reduction of O2. Mammalian complex IV is a large transmembrane protein with 6-13 subunits. The complex exists as a dimer. Subunits I and II contains all four redox centers (two hemes (a and a3) changing between Fe++ and Fe+++ and two Cu atoms (Cua and Cub) that vary between Cu+ and Cu++. Reductions of O2 to 2H2O takes place on the cytochrome a3-Cub complex.