Biochemistry I Oxidative Phosphorylation I. MITOCHONDRIA Mitochondria are cellular organelles which have two bilayer membranes, and two compartments defined by those membranes. Mitochondria are completely responsible for the production of ATP in chemotrophs (and even serve this purpose in phototrophs when light is limited). The first bilayer, the outer membrane, defines the boundaries of the organelle; it is about 70% protein by mass. It is very porous but even so, contains a transmembrane channel called porin. This protein allows the passage of molecules up to about 10,000 daltons or so to travel back and forth across the outer membrane; the outer membrane is fairly porous. Inside of the outer membrane is another membrane called the inner membrane. The inner membrane has a higher protein content, almost no cholesterol, and a higher proportion of unsaturated lipids. Almost nothing can travel across the inner membrane without the assistance of transport proteins (except water); its virtually impermeable. It is loaded with transport proteins and enzymes for metabolism. In between the outer and inner membrane is a space called the intermembrane space. Because the outer membrane is so porous, the intermembrane space and the cytoplasm are virtually homogeneous. Inside the inner membrane is an area called the matrix. The TCA Cycle takes place in the matrix on soluble and membrane bound enzymes. Recall that the primary product of the Krebs Cycle, with respect to energy, is NADH. NADH carries the free energy produced as a result - of catabolic processes, like glycolysis and the Krebs cycle, as hydride ions (H ). This free energy is used to regenerate ATP in the matrix. II. OXIDATION AND REDUCTION The regeneration of ATP depends upon the transfer of free energy in the form of hydride ions; high, energy electrons. NAD and FAD pick up the electrons in catabolic reactions of glycolysis and the TCA cycle. They then carry the electrons where they are used to drive endergonic processes. In a chemical reaction, if one substance loses electrons and another substance gains electrons, that chemical reaction is called an oxidation-reduction (red-ox) reaction: H 2(g) ½O 2(g) -> H2O(l) This is a red-ox reaction because electrons are transferred. The oxidation state of hydrogen goes from 0 to 1, its oxidations state increases; hydrogen is oxidized. The oxidation state of oxygen goes from 0 to -2, its oxidation state is reduced; oxygen is reduced. Since hydrogen is reduced and oxygen is oxidized, electrons are transferred from hydrogen to oxygen (electrons are transferred from the oxidized substance to the reduced substance). Likewise, NADH carries electrons to endergonic processes: NADH(aq) X (aq) -> NAD (aq) XH(aq) NADH loses electrons so it is oxidized. The electron acceptor, X, gets the electrons so it is reduced. NADH is a reducing agent. Reduction Potentials The ability of one substance to reduce another substance (transfer electrons to another
substance) is called its reduction potential,. For example, the reaction for the transfer of 2 electrons from NADH to oxygen and hydrogen ion is: NADH(aq) H (aq) ½O (aq) -> NAD (aq) H O(l) 2 2 the reduction potential for the process,, is 1.136V The relation between the standard free energy of a process and its reduction potential is given by: G = -n where n is the number of electrons transferred, is Faraday s constant, ~96.5kJ/V Notice that large positive reduction potentials lead to large negative standard free energy changes. So you see where the energy of the electrons is used; its freed by the electron transfer. The standard free energy change for the transfer of 2e- from NADH to oxygen is about -219kJ per mole of NADH; quite a bit of free energy. So what does the reduction potential tell you: the larger the reduction potential, the greater the probability that free energy will be generated when electrons are transferred to the acceptor (the greater the likely hood that the transfer will be spontaneous). As with other state functions, reversing the reaction reverses the reduction potential. Consider the same reaction reversed: NAD (aq) H O(l) -> NADH(aq) H (aq) ½O (aq) 2 2 = -1.136V The standard free energy change for the transfer of 2e- from water to NADH is about 219kJ per mole of water. This reaction has almost no chance of being spontaneous: - G = RTln([NADH][H ][O ] /[NAD ]) ½ 2 at normal body temperature, the ratio of product to reactant would have to be less than ~0.919 for the transfer to be spontaneous Dinucleotides in Metabolism There are primarily two dinucleotide coenzymes involved in the regeneration of ATP: NAD and FAD. The role of each is the same: Catabolism of organic material results in the release of large amount of free energy. This free energy is carried by electrons. The electrons are carried by the dinucleotides as hydride ions. NAD 2H 2e -> NADH H - - FAD 2H 2e -> FADH shows how NAD is reduced to NADH when it catches electrons. shows how FAD is reduced to FADH when it catches electrons. 2 2 Neither of these is a favorable reaction (they both have negative reduction potentials), but the free energy discharge resulting from catabolism drives the transfer of electrons to these carriers (one of the things accomplished by catabolism is loading these molecules up with electrons). The electrons they carry are the fuel that drives the re-attachment of phosphate to ADP (ATP synthesis/regeneration). So note, if loading the molecules with electrons requires free energy, unloading the electrons results in the release of free energy! III. OXIDATIVE PHOSPHORYLATION The primary product of the Krebs Cycle (and thus the catabolism of monosaccharides and other such carbon compounds), is NADH. The standard reduction potential for NAD is -0.32V. This means that this redox pair will clearly favor the oxidation of NADH as opposed to the reduction of NAD ; NADH carries high energy electrons that it wants to dump somewhere. When the electrons are dumped, they are accompanied with a good deal of free energy. That free energy is used to produce ATP from ADP; to attach phosphate groups to ADP molecules. ATP synthesis is called oxidative phosphorylation because electrons carries are oxidized so that ADP may be phosphorylated. So, what exactly do those electrons transferred do?
Electron Transport and Hydrogen Pumping First recall that the inner membrane is virtually impermeable to everything except water and that the intermembrane space is virtually the same as the cytoplasm because the outer membrane is so porus. Electron carriers deliver electrons to membrane proteins of the mitochondrial inner membrane. These proteins are electron transport machines; they transfer electrons to other electron transport machines until the electrons eventually end up transferred to oxygen and hydrogen ion (and water is made). Remember, these are high energy electrons, they carry a boatload of free energy. When they are transferred, the free energy is used to cause the electron transport proteins to pump hydrogen from inside the matrix into the intermembrane space. Why is energy needed for this? One, the innermembrane is not permeable to H. And two, even if it was, pumping H out creates an imbalance of H. The free energy is needed to push H out into i) a larger H concentration, and ii) a positive environment. Part of the free energy of the electrons is used to create a concentration/charge imbalance (gradient) of H. Each time electrons are transferred to a new electron transporter, several hydrogen ions are pumped out of the matrix. This is done about three times; one pair of electrons can supply the energy to expel about 10 H. The end result of this is to create a situation where there s a ton of hydrogen ion and positive charge outside an area that has less hydrogen ion and a more negative charge. Consequently the hydrogen ion outside of the matrix is extremely attracted to the matrix, however, the membrane is impermeable to hydrogen ion, so that attraction is potential energy! Part of the free energy from the electron transfer is converted into potential energy of attraction. Although the inner membrane is impermeable to H, there is a way for the H to return; a protein complex called ATP synthetase. So you got all these hydrogen ions outside the matrix with all that energy of attraction to the inside of the matrix. If they find a way in, there ll be a whole lot of free energy released on their re-entry. And that s just what ATP synthetase uses. It takes the free energy of the H returning into the matrix and uses it to attach phosphate to ADP! ATP synthesis is coupled to a type of osmosis, which is driven by electron transport.
Mechanics of Electron Transfer Each time something picks up electrons, it is reduced. Most of these such transfers occur using iron-sulfur (Fe-S) centers, cytochromes, copper ion, flavoproteins, and coenzyme Q. For instance, if electrons are transferred to an Fe-S center: Fe e -> Fe 3-2 the change in oxidation states causes conformational changes that result in H expulsion from the matrix. Osmosis without ATP Synthesis If H re-enters the mitochondria, somehow, without ATP synthesis, the free energy is released as thermal energy; heat. In fact, that is a mechanism many organisms use to keep warm. Hydrogen ion enters the matrix through special membrane proteins not linked to ATP synthesis. IV. USING ATP ATP/ADP Transport The ATP produced in the mitochondria is transported into the cytoplasm by ATP/ADP transport proteins. These membrane proteins transport an ADP into the matrix only when there is an ATP to transport out (once in the intermembrane space, each gets to or from the cytoplasm through porin in the outer membrane). Once in the cytoplasm, the ATP is distributed and used as needed. Quantitative Aspects of ATP Hydrolysis Whenever an there is a need for free energy, ATP is hydrolyzed according to the reaction: ATP(aq) H2O(l) ADP(aq) Pi(aq) 2 G ~ -30.5kJ/mol, 37 C, ph=7.0, [Mg ]~5mM 2 Normal conditions for a typical human red blood cell is 37 C, ph=7.0, [Mg ]~5mM, [ATP]~2.25mM, [ADP]~0.25mM, [P i]~1.65mm. The free energy change ( G) for the hydrolysis of 4 ATP under such conditions is: G = -5.27x10 kj/mol (about 53,000J of energy released for every mole of ATP hydrolyzed). The act of hoisting a one-pound weight in the air (about 5 feet) requires the expenditure of about 2 2 PE = gmh = 9.8m /s (0.455kg)(1.524m) ~ 6.8J The amount of ATP you d have to hydrolyze to complete that task is 6.8J/x = 53,000/1mol -4 x ~ 1.3x10 moles of ATP (about 130 mol of ATP) Each glucose completing glycolysis, the Kreb Cycle, and oxidative phosphorylation regenerates about 31 molecules of ATP. So you d need about 4 moles of glucose to raise a 1.0 pound weight 5 feet in the air. A typical candy bar contains about 100,000 moles of glucose (equivalents), so only about one-twenty-five thousandth of a candy bar would be required for that task.
SIDE NOTE A device for measuring reduction potential is a potentiometer. This device will measure the flow of electrons from one process to another. Two solutions, A and B, are connected by a wire and an agar bridge. The agar bridge will allow the passage of small ions and the wire will allow the flow of electrons. As shown, solution A is connected to the wiring on the left of a voltmeter (the side with the negative numbers) and solution B is connected to the wiring on the right side of the voltmeter (the side with the positive numbers). The direction of the electron flow will be opposite the needle deflection. For instance, if electrons flow from A to B (left to right), the needle will deflect left of zero toward the negative numbers (solution A is oxidized, solution B is reduced). If electrons flow from B to A (right to left), the needle will deflect right of zero toward the positive numbers (solution A is reduced, solution B is oxidized). In these devices, there will usually be a standard reaction on the positive side. A typical standard is a solution of 1M H (ph=0) and 1.0 atm H 2 called a hydrogen reference half-cell. Any solution connected to the reference cell that registers a negative voltage is sending electrons to the reference cell (hydrogen ion is reduced to molecular hydrogen gas). On the other hand, if a positive voltage is registered, the reference is sending electrons to the other solution, causing one of its components to be reduced. Solutions with a voltage less than zero are better at causing reduction (they undergo oxidation yielding electrons that reduce hydrogen ion; they re better reducing agents). Solutions with a voltage greater than zero are better at causing oxidation (they undergo reduction consuming electrons from the oxidation of molecular hydrogen gas; they re better oxidizing agents). The voltage registered is the reduction potential.
Spontaneity and Reduction Potential The spontaneity of a process is judged by its standard free energy change, G. A negative standard free energy change means spontaneity is not difficult to achieve. On the other hand, a positive standard free energy change means spontaneity will be difficult to achieve. As you should know, the relation between the standard free energy change, and spontaneity (the free energy change) is: G = G RTln([P]/[R]) where [P]=product and [R]=reactant; if P/R > 1 you re adding to G ; if P/R < 1, you re subtracting from G Adding to G makes G more positive; less spontaneous. Subtracting from G makes G more negative; more spontaneous. Reduction potential is a gauge of how easy it would be for some entity to accept electrons; to be reduced. It is related to the standard free energy change: G = -n ' where n is the number of electrons transferred, is Faraday s constant, ~96.5kJ/V, and ' is the reduction potential If the reduction potential is negative, G will be positive and the only way the transfer will be spontaneous is if P/R < 1 and the absolute value of RTln([P]/[R]) > G. If the reduction potential is positive, G will be negative and the transfer will be spontaneous as long as the absolute value of RTln([P]/[R]) < G. CASE 1 - NAD is reduced when it picks up electrons: NAD 2H 2e -> NADH H For this half-reaction (we don t show where the electrons come from, only where they re going, so it s a half-reaction); ' = -0.32V. The reduction potential is negative, so the standard free energy change will be positive and the transfer of electrons to NAD will not tend to take place spontaneously. - Reverse the reaction and we have NADH being oxidized: NADH H -> NAD 2H 2e For this half-reaction (we don t show where the electrons are going, only where they come from, so it s a half-reaction); ' = 0.32V. The reduction potential is positive so the standard free energy change will be negative and the transfer of electrons to NAD will tend to take place spontaneously. What does the reduction potential tell us about this reaction? That its easier to oxidized NADH than to reduce NAD! CASE 2 Consider a reaction that is not a half-reaction (we know the electrons come from NADH and go to oxygen); the reduction of oxygen to water using NADH: NADH H ½O 2 -> NAD H2O ' = 1.14V This reaction should tend toward spontaneous; the reduction potential is positive so the standard free energy change should be negative. CASE 3 If you know the reduction potentials for different reactions, you can determine the reduction potential for the reactions combined the same way we use Hess s Law. You just have to decide which way you want the electrons transferred. - FAD 2H 2e -> FADH2 ' = -0.210V 3-2 Fe e -> Fe ' = 0.771V 2 Suppose we want to transfer electrons from Fe to FAD. We d have to rearrange the iron reaction, then add the resulting reactions to get the reduction potential: - FAD 2H 2e -> FADH2 ' = -0.210V 2 3-2Fe -> 2Fe 2e ' = -1.542V =================================================== 2 3 FAD 2H 2Fe -> FADH 2Fe ' = -1.752V 2 The reduction potential for this reaction is a large negative number. There is almost no chance that iron(ii) can transfer electrons to FAD without a good deal of energy input. There is very little chance it ll happen spontaneously.