Krebs Cycle and Electron Transport Chain

The next sequence in the transport chain involves cytochromes, iron-sulfur clusters, and copper atoms located between coenzyme Q and molecular oxygen. Electrons are passed successively from coenzyme Q to cytochrome b, to Fe-S, to cytochrome c1, to cytochrome c, to Cu, to cytochrome a, and finnally to cytochrome a3. Each carrier in the chain is reduced as it picks up electrons and is oxidized as it gives up electrons. The last cytochrome, cytochrome a3, passes its electrons to one-half of a molecule of oxygen, which becomes negatively charged and then picks up 2H+ from the surrounding medium to form H2O. This is the only point in aerobic cellular respiration where O2 is consumed. 

Complex I:

matrix: 2H+ reduced (from NADH + H+) + 2H+ pumped out

IMS: 4H+ pumped in

Complex II:

matrix: 2H+ reduced (from succinate)

IMS: 0H+ pumped in

Complex III:

matrix: 2H+ pumped out (when not considering complex II, you'd count 4H+ here)

IMS: 4H+ pumped in (2 from complex II)

Complex IV:

matrix: 2H+ reduced (to H2O, we won't consider it as you said) + 2H+ pumped out

IMS: 2H+ pumped in

TOTAL:

matrix: 10H+ subtracted/pumped out (with 2H+ to water, total 12H+)

IMS: 10H+ pumped in

Note that FADH2, derived from the citric acid cycle, is another source of electrons. However, FADH2 adds its electrons to the electron transport chain at a lower energy level than does NADH+H+. Because of this, the electron transport chain produces about one-third less energy for ATP generation when FADH2 donates electrons as compared with NADH+H+, so think of the same proton argument above, but entering at complex III so only 6H+

Oxidative Phosphorylation

The thermodynamic view of chemiosmotic coupling

How much energy is stored in the electrochemical H+ gradient across the inner mitochondrial membrane?

• The proton gradient sets up both a concentration gradient, and an electrostatic gradient. The free energy term relating to concentration and charge differences across a membrane is as follows:

DG = RTln[C2]/[C1] + ZᵹΔΨ 

• RTln[C2]/[C1] is the contribution of the concentration gradient to the free energy term. C2 would be the proton concentration "outside" the membrane (i.e. on the cytosolic side) and C1 would be the concentration of protons in the matrix. It is easier to work in pH units (i.e. -log[H+]), in which case the above equation becomes:

ΔG = 2.30*RT(log[C2] - log[C1]) + ZᵹΔΨ 

ΔG = 2.30*RT(-pH[out] + pH[in]) + ZᵹΔΨ 

ΔG = -2.30*RT(pH[out] - pH[in]) + ZᵹΔΨ 

The DpH value across the mitochondrial membrane is on the order of 1.0 pH unit lower outside than inside (protons pumped out, therefore pH is higher inside), therefore the above equation reduces to:

ΔG = -2.30*RT(-1) + ZᵹΔΨ 

ΔG = 2.30*RT + ZᵹΔΨ 

• The ZᵹΔΨ  term refers to the contribution of the electrostatic gradient across the membrane, where Z is the charge on the proton (+1), ᵹ is the Faraday constant (96.5 kJ/volt mol) and ΔΨ is the potential difference across the membrane (typically measured to be around 0.18V (positive outside, negative inside the matrix). Thus, the above equation reduces to:

ΔG = 2.30*RT + (1)*(96.5kJ/volt mol)*(0.18volt)

ΔG = 2.30*(8.31J/K mol)(310 K) + (96.5 kJ/volt mol)(0.18 volt)

ΔG = 5.93 kJ/mol + 17.4 kJ/mol

ΔG = 23.3 kJ/mol

Conclusions from this analysis:

• The free energy change is unfavorable for both the concentration term and the electrostatic term. In other words, there are more protons outside, so it will take energy to pump a proton out, and also that since the outside is more positively charged compared with inside, it is energetically costly to move a positively charged ion (i.e. proton) from inside to outside

• The term with the largest magnitude is the electrostatic term. Thus, while the concentration gradient is unfavorable, it really is the charge across the membrane that represents the greatest amount of stored energy

• Consequently, the inward flow of protons will release an amount of energy equal to DG = -23.3 kJ/mol. It is this energy release that is coupled to the production of ATP

 ATP Synthase

The molecular machine that carries out ATP synthesis in the mitochondrion is called ATP synthase, or F1F0-ATPase. This is a pretty large molecular complex that is actually visible as "bumps" on the inner mitochondrial matrix (on the matrix side).

ATP synthase consists of two molecular complexes, termed the F1 and F0 complex

• The F0 complex is membrane-bound and contains three subunits (a, b, and c). It contains a transmembrane pore that allows passage of protons from the intermembrane space into the matrix

• The F1 complex is associated with the F0 complex, but protrudes into the matrix environment (i.e. it is the "bump" seen on electromicrographs). It contains five subunits a, b, g, d, and e. The F1 complex contains the ATP synthesis activity (located within each of the three b subunits).

The ATP synthase complex is a molecular motor!

The c/g/e assembly rotates in relationship to the a/b/d/a/b complex (which is rigid)

• Proton transport from the intermembrane space into the matrix region (driven by chemiosmotic force), through the proton pore in the c complex, drives the motor.

• The rotation of the g subunit in contact with the a/b subunits confers sequential conformational changes upon the a/b complex which results in ATP synthesis activity within the b subunit

• The c/g/e complex that rotates is often called the "rotor", and the stationary a/b/d/a/b complex is termed the "stator" (in relationship to similar parts in an electric motor)

The three a/b complexes are identical to each other, but are able to exist in three different conformational states:

1. The "open" conformation (conformation O). Low affinity for ATP, ADP or phosphate ion, and is catalytically inactive

2. The "loose" conformation (conformation L). Binds ATP, ADP and phosphate ion loosely; no catalytic activity

3. The "tight" conformation (conformation T). Binds ATP, ADP and phosphate tightly, is catalytically active

The conformational states of the three a/b complexes in the "stator" are influenced by asymmetric interactions with the "rotor" and sequentially cycle through these states:

 The transport of about 3 protons is required for the synthesis of one ATP

 Inhibitors of electron transport complexes block electron transport

Inhibitors of complex I:

• Rotenone. Found in roots of certain plants. Will kill insects or animals that eat the root!

• Mercury compounds.

• Barbiturates

• Demerol

These compounds all inhibit the reduction of coenzyme Q and the oxidation of the Fe-S clusters of NADH-ubiquinone reductase

 Inhibitors of complex II:

• Carboxin. Carboxin is a systemic anilide fungicide. It is used as a seed treatment for control of smut, rot, and blight on barley, oats, rice, cotton, vegetables, corn and wheat. Inhibits succinate-UQ reductase of complex II.

Inhibitors of complex III:

• Antimycin. An antibiotic produced by Streptomyces griseus

• Thenoyltrifluoroacetone. Commonly used as an extraction reagent in organic chemistry (this may explain the behavior of some organic chemists...)

These all inhibit the ubiquinone-cytochrome c reductase by blocking the electron transfer between bH and coenzyme Q in the QN site.

 Inhibitors of complex IV:

• Cyanide. Used by the state to eliminate bad people.

• Azide. Used as anti-microbial preservative (but not in foods!)

• Carbon monoxide. Product of incomplete combustion of fossil fuels (as occurs when people use fires in a confined area to keep warm)

Cyanide and azide bind to the Fe3+ form of cytochrome a3. Carbon monoxide binds to the Fe2+ form. Heme in red blood cells also contains the Fe2+ form of iron, and there is a lot of heme in your body. This means that you need a lot more carbon monoxide to kill you than cyanide.

 Inhibitors of ATP synthase:

• Oligomycin. Produced by bacteria (actinomycetes) and is a potent anti-fungal agent

• DCCD. Dicyclohexylcarbodiimide. Used in peptide synthesis and also as an elastomer reagent in synthetic and natural rubber.

Oligomycin blocks the movement of protons through the F0 complex. DCCD bonds to carboxyl groups on acidic side chains in proteins, including one in the c subunit of F0 complex and mucks up the proton channel.

 "Uncouplers"

 Uncouplers act by dissipating the proton gradient across the inner mitochondrial membrane. As a class of molecules they share two common structural features:

1. They are hydrophobic

2. They have a functional group that can reversibly protonated

By virtue of their hydrophobic property they can insert into bilayer membranes (e.g. the inner mitochondrial membrane). Since they contain a group that can be reversibly protonated, they can transport protons across the membrane. Thus, they are involved in the facilitated diffusion of protons across the inner mitochondrial membrane (from high to low concentration) and they destroy the gradient. What will be the result of destroying the proton gradient?

• Energy released in electron transport and oxidative phosphorylation will not be coupled to ATP synthesis, instead, it will essentially be released as heat

• This is a mechanism by which organisms can use the energy stored in glucose to generate heat

Examples of uncoupled oxidative phosphorylation for heat generation:

• Newborns (lose a lot of heat due to high surface area/volume)

• Hibernating animals (dang its cold...)

• Certain cold-adapted animals

• Some plants actually use this to disperse volatile components

• "Brown fat cells" contain a lot of mitochondria and is used in cold adapted animals for heat generation in uncoupled oxidative phosphorylation

 ATP produced in the mitochondria exits via an ATP-ADP translocase

Once it is made, ATP in the mitochondria must be transported out for use in other parts of the cell. Likewise, ADP must be transported into the mitochondria to regenerate ATP (there is a large ATP demand, but a relatively low concentration, so demand is met by continual recycling of the ATP pool)

• ATP within the mitochondria is made within the matrix.

• ATP and ADP are charged molecules than cannot readily pass across lipid bilayers.

• The inner membrane does not have porins and is not freely permeable like the outer membrane of the mitochondria

Transport of ATP out of, and ADP into, the mitochondrial matrix is controlled by the ATP-ADP translocase membrane protein.

• ATP and ADP transport is tightly coupled by this membrane protein. One molecule of ADP is transported into the matrix for every molecule of ATP that is transported out.

 The charge on ATP(4-) compared to the charge on ADP(3-), and the proton gradient across the inner membrane, favors the spontaneous transport of ATP out of, and ADP into, the matrix:

The passage of ATP out of the matrix, and ADP into the matrix, decreases the charge across the inner membrane by -1 units (i.e. it is as if we have removed a proton from outside the matrix). Thus, four protons are required to make an ATP - three for synthesis plus one that is "neutralized" upon export of the ATP.

 The P/O Ratio

The P/O ratio refers to the number of molecules of ATP formed in oxidative phosphorylation for every two electrons flowing through a defined segment of the electron transport chain. There is still some debate regarding this value. One such estimate is as follows:

• 10H+ are transported out of the matrix for every two electrons that pass from NADH to O2

• 4 H+ are transported into the matrix for every ATP synthesized

• P/O = (1 ATP/4H+)*(10H+/NADH)*(1 NADH/2e') = 2.5 ATP/2e'

 This is more or less close to the 3 ATP's for each NADH that enters oxidative phosphorylation quoted previously.

 Shuttle systems feed the electrons of cytosolic NADH into electron transport

NADH is produced in the cytosol by glycolysis (by glyceraldehyde-3-phosphate dehydrogenase):

• If this NADH were not subsequently oxidized to NAD+, glycolysis would come to a screetching halt as NAD+ ran out.

• Eukaryotic systems have several shuttle systems that "harvest" electrons from NADH in the cytosol, and transport these electrons to the mitochondria for passage down the electron transport system (and in doing so, regenerate NAD+ in the cytosol)

The glycerophosphate shuttle

• The electrons in NADH are transferred to DHAP to produce glycerol-3-phosphate and NAD+.

• The Glycerol-3-phosphate can transfer its electrons directly to FAD in the inner mitochondrial membrane (producing FADH2)

• The FADH2 can proceed through the electron transport chain, put produces fewer ATP molecules compared to NADH passing through the chein (making 1.5 - 2 ATP's)

The malate-aspartate shuttle

The malate-aspartate shuttle oxidizes NADH in the cytosol by reducing oxaloacetate to malate. Malate is transported into the matrix by the a-ketoglutarate-malate transporter. In the matrix malate is oxidized to oxaloacetate and this is transported back out, after first being transaminated to aspartate, by the asparate-glutamate carrier. Glutamate and a-ketoglutarate are coordinately transported across the mitochondrial membrane in the opposite direction by the same carriers (for a zero net charge transport):

 Final note on glycolysis, TCA cycle and electron transport

Combustion of glucose has an associated free energy change of -2937 kJ/mol. Glycolysis, the TCA cycle and electron transport (oxidative phosphorylation) is able to produce between 32-38 ATP's per mole. If one assumes that ATP hydrolysis (under cellular concentration conditions) yields -50 kJ/mol per ATP, the energy captured is between 1600 - 1900 kJ/mol or 54-65%. Pretty amazing when you consider that some of the "non-captured" energy is heat, and this is used to keep us warm, the efficiency is dramatic.