Lectures 6 and 7

Details of Chemiosmotic Theory
When first proposed there was little scientific evidence. The theory met with considerable opposition. We can summarize the evidence for the theory as follows:-

1a. Demonstrated linkage between proton pumping and electron transport
How?

1. Using mitrochondria starved of O₂.
2. Resupply a burst of O₂ and measure pH changes. pH dropped and gradually recovered.
3. Δ pH (NADH) > Δ pH (succinate)
   Δ ATP (NADH) > Δ ATP (succinate)

   

4. Oxygen consumption is a measure of electron (e^-) flow.
   Δ pH is a measure of proton pumping.
5. Stoichiometries first reported have since been altered. This is because there is buffering capacity within mitochondria making it difficult to obtain absolute values of ΔpH, and because some of proton gradient may be consumed by processes other than ATP synthesis. For example transport of various ions such as Ca²⁺, Pi
6. The source of electrons is typically NADH: NADH -----> NAD⁺ + H⁺ + 2e^-
   Early reports suggested:

   
   6 H⁺ pumped per NADH oxidized
   4 H⁺ pumped per FADH₂ oxidized
   Later values suggest higher values:
   10 H⁺ pumped per NADH oxidized
   6 H⁺ pumped per FADH₂ oxidized

7. Consistent with the early estimate of 6 H⁺ per NADH oxidized it was suggested that this pumping of H⁺ occurred at 3 sites, namely through complex I, complex III and complex IV. Now we are more conservative and suggest the values may be between 3 and 4 at each of three sites.

Electron Flow/Proton Pumping/ATP synthesis
Not only was Δ pH created during electron flow (when oxygen was available) but measurements of ATP synthesis showed interesting correlations between electron flow and ATP synthesis.

See Figs 19-17 a and b in Lehninger.

Fig. 19-17a

1. When ADP and Pi was supplied in the presence of oxygen there was no ATP synthesized until succinate was added. Why?
2. When succinate was added both oxygen consumption and ATP synthesis increased. Why?
3. When CN^- (cyanide) was added both oxygen consumption and ATP synthesis ceased. Why?

Fig 19-17b

1. When succinate was added no oxygen was consumed in the absence of ADP + Pi. This is a very important observation. Can you explain it?
2. Oligomycin blocked oxygen consumption and ATP production. Why?
3. DNP (2, 4-Dinitrophenol) blocked ATP synthesis but not oxygen consumption. Can you explain?

Uncouplers
The DNP results provided proof that an impermeable membrane barrier is essential for generating ATP and this is consistent with the chemiosmotic theory. How does DNP dissipate the proton gradient? Being a phenol the phenolic hydroxide can dissociate and generate a phenoxide ion. This can pick up a proton and being lipid soluble the DNP can easily migrate across the membrane shuttling protons back and forth.

Evidence from other protonophores
A carrier of protons is referred to as a protonophore. DNP is an example of such a protonophore. Other protonophores include CCCP (carbonyl cyanide m-chlorophenylhydrazone) and the antibiotic peptide gramicidin. Gramicidin allows H⁺ or monovalent cations to pass down their electrochemical gradient. The general term for ion carriers of this sort is ionophore. A protonophore is one kind of ionophore.

Valinomycin is a K⁺ ionophore, that will enable K⁺ ions to traverse the membrane along the electrochemical gradient for K⁺. This may be due to a concentration difference for K⁺ (ΔK⁺) or due to an electrical potential difference. Thus if H⁺ ions are being pumped out by the etc and K⁺ is present together with valinomycin, the valinomycin will distribute itself in the mitochondrial membrane and by allowing K⁺ to move into the matrix along the electrical gradient due to ΔΨ, K⁺ will dissipate the ΔΨ created by H⁺ pumping.

Nigericin is another ionophore that permits neutral (1:1) K⁺/H⁺ exchange across a membrane. Hence unlike valinomycin that can dissipate ΔΨ, nigericin does not affect ΔΨ but it will replace ΔpH.

With respect to ATP synthesis the following ionophores have the effects shown. Can you explain the results?

Ionophore	ATP synthesis
Valinomycin	yes
Nigericin	yes
Valinomycin + Nigericin	no

Uncoupling ATP synthesis is not always bad
When electron flow occurs in uncoupled mitochondria, instead of making ATP, the proton gradient is dissipated and the free energy of redox reactions is converted to heat. In hibernating animals mitochondria in brown fat are deliberately uncoupled to maintain body temperature. Either special proteins or fatty acids are inserted into the mitochondrial membrane and act as uncouplers. One such protein called thermogenin allows exchange of OH^- with Cl^-. Skunk cabbage also converts redox energy to heat by using an "alternative pathway" of electron flow to oxygen. This pathway is called the cyanide-resistant pathway (because it is not blocked by cyanide) serves to generate heat and in colder regions the skunk cabbage may melt the snow around its leaves. The elevated temperatures of the flower volatilize odouriferous compounds that attract pollination insects because of the simulated smell of rotting material.

c. Thermodynamics of proton pumping
As a result of H⁺ pumping a steady value of Δ pH = 1.4 units and ΔΨ = 140mV (matrix negative) is achieved. We can ask the next question, namely, is this sufficient to overcome the Δ G⁰¹ or ΔG value for ATP synthesis at 25°C.
ΔG^OI = 2.303 RT log₁₀ [H⁺]_o/[H⁺]_i + zFΔΨ
ΔG^OI = -21.5 kJ mol^-1 protons
Assuming a value of 3ATP synthesized per NADH oxidized and the original value of 6 protons pumped per NADH oxidized gives a value of 1 ATP synthesized for every 2H⁺.

If ΔG^OI = -21.5 kJ mol^-1 protons
ΔG^OI = -43 kJ (2 mol)^-1 protons

This is sufficient energy to generate ATP, the typical value of ΔG^OI being ~ 30.5 kJ mol^-1.

Thus, thermodynamically the chemiosmotic theory passes the test. But so what? Just because there is a proton gradient and it has sufficient energy, does this prove the hypothesis?

Direct Evidence for ATP synthesis via the p.m.f.
The first such evidence, referred to as "acid-bath experiments" came from studies of ATP synthesis by chloroplasts in the dark (Andre Jagendorf, 1966).

Similar experiments were carried out soon after using mitochondria.

1. Mitochondria were incubated at pH9 in 100 mM KCl.
2. Mitochondria were then resuspended in media at pH7 containing no KCl. ADP plus Pi were provided together with the ionophore valinomycin, but no oxidizable substrates for e^- transport.
3. ATP was synthesized

Can you interpret these results?

d. Asymmetric Organization of redox Carriers
If the proton pumping of the redox carriers were in random directions (out of and into the matrix) there would be no net p.m.f. generated. Therefore the carriers must be placed asymmetrically in the inner membrane of the mitochondrion. Experiments with antibodies, proteolytic enzymes and non-penetrating labels confirm that some of the respiratory complexes (I, II, III and IV) face one side, others face the opposite side and some are transmembrane.

One example of a non-penetrating label is lactoperoxidose. When a membrane is treated with this enzyme together with ¹²⁵I, only exposed proteins are labeled. Thus we can determine which polypeptides face towards the outside. However, we can place mitochondria in hypotonic solution and make them swell and burst. Then interior proteins can also be labeled.

e. Reconstituted Systems

Efraim Racker (1960's) achieved successful disassembly and reconstitution of mitochondrial electron transport and ATP synthesis.

1. Take intact mitochondria.
2. Disrupt by osmotic rupture, or by detergent treatment; sub-mitochondrial vesicles are generated with tiny stalked particles facing outward. These are called F₁ particles.
3. These sub-mitochondrial vesicles are able to conduct redox reactions and make ATP.
4. Disruption of the vesicles by urea or by shaking with glass beads removed the F₁ particles leaving smooth vesicles. Using centrifugation, Racker separated the F₁ particles from the vesicles.
5. The vesicles could still carry out redox reactions but could not make ATP. The F₁ particles could not carry out electron transport reactions nor could they make ATP, but they were capable of hydrolyzing ATP.
6. Mixing the two fractions (vesicles and F₁ particles) restored the capacity to carry out redox reactions and make ATP.
   Racker called the particles added back to the smooth vesicles the "coupling factor" because they restored ATP synthesis. He abbreviated this to F for factor. Hence the knob-like structures on a stalk were called F₁ particles. They are attached to the mitochondrial membrane by a stalk that fits into the Fo particle that is embedded in the mitochondrial membrane.
7. Another reconstituted system using rhodopsin and ATP synthetase is shown below:

   

f. Some Minor but Interesting Observations

1. If the redox complexes are solubilized (see Figure 19-17 (Lehninger), they are capable of redox reactions but unable to synthesize ATP.
2. The purified complexes can be reconstituted by dispersing these protein complexes in artificial phospholipd vesicles. If we include fluorescent dyes inside the vesicles that give out characteristic fluorescence as the pH changes, we can monitor the pH inside the vesicle.
3. Using each of Complex I, Complex III, Complex IV and the F₁Fo particles and dispersing each one of them separately in phospholipid vesicles, the pH went acid inside the vesicles when appropriate substrate was provided:

   Complex	Substrate	pH inside vesicle
   Complex I	NADH	acidified
   Complex III	Reduced ubiquinone/cytochrome c	acidified
   Complex IV	cytochrome c/O2	acidified
   F1Fo particles	ATP	acidified

   
   Can you predict the reactions for each of the above?

How does proton transport occur during redox reactions?

(mobile carriers and conformational changes)

You will notice that some of the redox participants of the ETC involve electron flow and proton exchange

e.g., QH₂ + 2 Cyt C_1(ox) -----> Q + 2H⁺ + 2 Cyt C_1(red)

Other redox reactions involve only electron transfers:

e.g. Cyt C_1(red) + Cu²⁺_A center -----> Cyt c_1(ox) + Cu⁺_A center
(see Fig 19-13)

When a reaction involving proton transfer is followed by a redox reaction involving only e^- transfer, the proton(s) are available for vectoral transport across the mitochondrial membrane. How does this occur?

Take the case of Ubiquinone (see Fig 19-2)

This molecule can go from the fully oxidized quinone structure to the fully reduced Ubiquinol by accepting 2H⁺ and 2e^- in two steps. The small ubiquinone is a lipid-soluble molecule and can readily traverse the membrane, releasing protons vectorially when the quinol form of ubiquinone reduces cytochrome C₁ (actually via the Fe-S protein shown in purple (Fig 19-11). Thus vectorial H⁺ pumping can be achieved due to the mobility of the proton carrier.

Complex 1 and 4 are not mobile carriers but they do pump protons too (Fig 19-9 and Fig 19-13). It is thought that the electron transfer to these complexes brings about conformational changes of the protein that are sufficient (energetically) to cause transfer of protons across the mitochondrial membrane.

Final point regarding the meaning of proton motive force

By analogy with electro motive force, the driving force for electron flow around an electrical circuit, Peter Mitchell suggested that we use the term proton motove force to describe the free energy for H⁺ across a membrane circuit. It is not actually the free energy but it is very close to that state variable. Clearly if it were identical to free energy there would seem no reason for a redundant term. Let me demonstrate the very subtle difference between the free energy difference for H⁺ and p.m.f.

Now ΔG^OI is measured in the classic way in kJoules per mol. Since we are discussing H⁺, we could represent the formula as follows :-

ΔG^OI = RTln [H⁺]_o/[H⁺]_i +zFΔΨ

First let us convert ln to log₁₀ by multiplying by 2.303

ΔG^OI = 2.303RTlog₁₀[H⁺]_o/[H⁺]_i +zFΔΨ

Since log [H⁺] = pH

ΔG^OI = 2.303RT(pH_o-pH_i) + zFΔΨ

ΔG^OI = 2.303RT(Δ pH_o-i) + zFΔΨ

If we now divide throughout by zF we convert kJ mol^-1 to V

ΔG^OI/zF = (2.303RT(ΔpH_o-i))/zF + ΔΨ

The term ΔG^OI/zF is the actual proton motive force

So for the mitochondrial membrane with ΔpH_o-i =-1.4 units and ΔΨ = -0.140 V

p.m.f. = (2.303 (8.315 x 298) x -1.4)/(+1 x 96480) + -0.140

p.m.f. = -0.22 V

To convert p.m.f. back to ΔG^OI

ΔG^OI = -zF(p.m.f)

In the above example:

ΔG^OI = +1 x 96480 (-0.22)

= -21.22 kJ mol^-1