Electron Transport System:The Chemiosmotic Theory, redox reactions of the

electron transport system, NAD/ATP exchange ratio

Bioc 460 Spring 2008 - Lecture 29 (Miesfeld)

Hydrogen cyanide is a deadly gas that blocks e- transport from

complex IV to O2 in the ETS

Rotenone (rat poison) blocks e- transport through FeS clusters

Passing the baton is analogous to passing

along the e- in the ETS

• The Electron Transport System converts redox energy into proton-motive force. In this “pathway” the oxidation of NADH and FADH2 is coupled to the reduction of O2 to form H2O. The proton motive force is used to induce conformational changes in the ATP synthase complex.

• The Chemiosmotic Theory states that energy from redox reactions is translated into vectorial energy by coupling electron transfer to membrane bound proton pumps that transverse a proton impermeable membrane and thereby establish an electrochemical proton gradient.

• The ATP currency exchange ratios of NADH and FADH2 reflect ATP synthesis in response to H+ movement through the ATP synthase complex. It takes 4 H+ to synthesize 1 ATP, and since NADH oxidation pumps across 10 H+, the exchange ratio is 2.5 ATP/NADH. However, FADH2 oxidation only results in 6 H+ being pumped across the membrane, and therefore the exchange ratio is 1.5 ATP/ FADH2.

Key Concepts in the Electron Transport System

The Electron Transport System (ETS) is intimately linked to the process of oxidative phosphorylation, both of which take place within the mitochondrial matrix.

Photosynthesis also uses a form of electron transport that is driven by light absorption rather than redox energy.

Peter Mitchell's Chemiosmotic Theory

• Oxidation of NADH and FADH2 in the mitochondrial matrix by the electron transport system links redox energy to ATP synthesis.

• Chemiosmosis involves the outward pumping of H+ from the mitochondrial matrix through three protein complexes in the electron transport system (ETS complexes I, III, IV).

• H+ flow back down the proton gradient through the membrane-bound ATP synthase complex in response to a chemical (H+ concentration) and electrical (separation of charge) differential.

Overview of Chemiosmotic Theory

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Electron TransportSystem

FADH2

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Energy Conversion Requires the Proton Circuit

Basic Components of the Chemiosmotic Theory

Basic Components of the Chemiosmotic Theory

• Energy from redox reactions or light is translated into vectorial energy and a proton circuit.

• Vectorial H+ pumping results in chemical gradient (pH) and a membrane potential ΔΨ (Δpsi)

• Separation of charge is due to build-up of positively-charged protons (H+) and negative hydroxyl ions (OH-)

Basic Components of the Chemiosmotic Theory

• In mitochondria, the contribution of ΔΨ (ΔV) to ΔG is actually greater than that of ΔpH (the ΔpH across the mitochondrial membrane is only 1 pH unit)

• In chloroplasts, the ΔpH contribution to ΔG is much more significant with ΔpH close to 3 pH units

• Change in free energy (ΔG) for a membrane transport process is the sum of the ion concentration (RT·ln(C2/C1)) and the membrane potential (ZFΔV)

• In mitochondria, the ZFΔV term makes a larger contribution than does RT·ln(C2/C1).

The Mitochondrion, the Powerhouse of the Cell

A critical feature of the mitochondrion is the extensive surface area of the inner mitochondrial membrane which forms the proton-impermeable barrier required for chemiosmosis.

Peter Mitchell - Eccentric Scholar

He established the Glynn Research Institute in the early 1960s with a research staff of less than twenty, and remained a private research institution for almost 30 years. Mitchell's uncle was Sir Godfrey Mitchell who owned George Wimpy and Company Limited, the largest construction company in England at the time.

How was Mitchell’s idea proven?

Using biochemical approaches:

1. "inside-out" submitochondrial membrane vesicles that could be shown to pump protons into the interior of the vesicle when oxidizable substrate was made available.

2. artificial vesicles containing bacterial rhodopsin protein were exposed to light• proton pumping by the

bacteriorhodopsin protein resulted in both inward proton pumping

• ATP synthesis on the vesicle surface

The Nobel Prize in Chemistry 1978

Peter Mitchell's speech at the Nobel Banquet, December 10, 1978:

The philosopher Karl Popper, the economist F. A. Hayek, and the art historian K. H. Gombrich have shown that the creative process in science and art consists of two main activities: an imaginative jumping forward to a new abstraction or simplified representation, followed by a critical looking back to see how nature appears in the light of the new vision. The imaginative leap forward is a hazardous, unreasonable activity. Reason can be used only when looking critically back. Moreover, in the experimental sciences, the scientific fraternity must test a new theory to destruction, if possible. Meanwhile, the originator of a theory may have a very lonely time, especially if his colleagues find his views of nature unfamiliar, and difficult to appreciate.

Pathway Questions

1. What does the electron transport system/oxidative phosphorylation accomplish for the cell?

– Generates ATP derived from oxidation of metabolic fuels accounting for 28 out of 32 ATP (88%) obtained from glucose catabolism.

– Tissue-specific expression of uncoupling protein-1 (UCP1) in brown adipose tissue of mammals short-circuits the electron transport system and thereby produces heat for thermoregulation.

2. What is the overall net reaction of NADH oxidation by the coupled electron transport and oxidative phosphorylation pathway?

2 NADH + 2 H+ + 5 ADP + 5 Pi + O2 → 2 NAD+ + 5 ATP +2 H2O

Pathway Questions3. What are the key enzymes in the electron transport and oxidative

phosphorylation pathway?

ATP synthase complex – the enzyme responsible for converting proton-motive force (energy available from the electrochemical proton gradient) into net ATP synthesis through a series of proton-driven conformational changes.

NADH dehydrogenase – also called complex I or NADH-ubiquinone oxidoreductase. This enzyme catalyzes the first redox reaction in the electron transport system in which NADH oxidation is coupled to FMN reduction and pumps 4 H+ into the inter-membrane space.

Ubiquinone-cytochrome c oxidoreductase - also called complex III, translocates 4 H+ across the membrane via the Q cycle and has the important role of facilitating electron transfer from a two electron carrier (QH2), to cytochrome c, a mobile protein carrier that transfers one electron at a time to complex IV.

Cytochrome c oxidase - also called complex IV pumps 2 H+ into the inter-membrane space and catalyzes the last redox reaction in the electron transport system in which cytochrome a3 oxidation is coupled to the reduction of molecular oxygen to form water ( O2 + 2 e- + 2 H+ → H2O).

Pathway Questions

4. What are examples of the electron transport system and oxidative phosphorylation?

Cyanide binds to the heme group in cytochrome a3 of complex IV and blocks the electron transport system by preventing the reduction of oxygen to form H2O. Hydrogen cyanide gas is the lethal compound produced in prison gas chambers when sodium cyanide crystals are dropped into sulfuric acid.

The Electron Transport System Is A Series Of Coupled Redox Reactions

The electron transport system consists of five large protein complexes:

1. Complex I; NADH-ubiquinone oxidoreductase (NADH dehydrogenase

2. Complex II; succinate dehydrogenase (citrate cycle enzyme

3. Complex III; Ubiquinone-cytochrome c oxidoreductase4. Complex IV; cytochrome c oxidase

5. F1F0 ATP synthase complex consisting of a "stalk" (F0) and a spherical "head" (F1)

It was possible to order the four electron transport system complexes because of:

• Specific redox reaction inhibitors(such as rotenone, antimycin A and cyanide)

• Known reduction potentials (Eº') of conjugate redox pairs

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The stoichiometry of "proton pumping" is:

4 H+ in complex I4 H+ in complex III2 H+ in complex IV

(10 H+/NADH and 6 H+/FADH2)

FADH2

Metabolic Fuel for Electron Transport

• NADH and FADH2 feed into the electron transport system from the citrate cycle and fatty acid oxidation pathways.

• Pairs of electrons (2 e-) are donated by NADH and FADH2 to complex I and II, respectively

• Pairs of electrons flow through the electron transport system until they are used to reduce oxygen to form water (O2 + 2 e- + 2 H+ → H2O).

• The two mobile electron carriers in this series of reactions are coenzyme Q (Q), also called ubiquinone, and cytochrome c which transfer electrons between various complexes.

How is the energy released by redox reactions used to "pump" protons into the inter-membrane space?

• a redox loop mechanism – Q cycle in complex III

• redox-driven conformational changes : “proton pump”– complexes I and IV

Separation of the H+ and e- on opposite sides of the membrane

The Q cycle in complex III uses this mechanism to translocate protons across the membrane

Redox-driven conformational changes in the protein complex "pump" protons across the membrane by altering pKa values of functional groups located on the inner and outer faces of the membrane.

Complex I: NADH-ubiquinone oxidoreductase

Complex 1 passes along 2 e- obtained from the oxidation of NADH to Q using a coupled reaction mechanism that results in the net movement of 4 H+ across the membrane.

Complex II: Succinate dehydrogenase

The 2 e- extracted from succinate in the citrate cycle is passed through the other protein subunits in the complex to Q as shown below. No protons are translocated across the inner mitochondrial membrane by complex II.

Complex III: Ubiquinone-cytochrome c oxidoreductase

Note the relative position of the electron carriers and the presence of two distinct binding sites for ubiquinone called QP and QN, which play a crucial role in diverting one electron at a time to cytochrome c via the Q cycle.

The terms QP and QN refer to the proximity of the sites to the positive (inter-membrane space) and negative (matrix) sides of the membrane.

The Q Cycle

• Functions as both a mobile electron carrier and a "transformer" that converts the 2 e- transport system used by complexes I and II, into a 1 e- transport system required by cytochrome C.

• The Q cycle requires that 2 QH2 molecules get oxidized by complex III, with one of QH2 molecule being re-formed by reduction to give a net oxidation of 1 QH2 molecule.

Four Steps of the Q Cycle

Converts a 2 e- carrier (QH2) into a 1 e- carrier (cytochrome c).

This requires that 2 QH2 molecules be oxidized, with 1 QH2 being reformed.

Note that the Q cycle reactions require that 2H+ from the matrix be used to regenerate QH2, even though 4H+ are translocated.

However, this apparent imbalance of 2H+ is corrected by the redox reactions of complex IV where 2H+ are required to reduce oxygen to water and 2H+ are pumped across the membrane.

Therefore, the net translocation of protons across the membrane in the combined redox reactions of complexes III and IV becomes 6 H+N → 6 H+P.

2 H+N

2 H+P

2 H+P

To see how the Q cycle accomplishes the 2 e- → 1 e- + 1 e- conversion process, write out two separate QH2 oxidation reactions and then sum them to get the net reaction for complex III:

QH2 + Cyt c (oxidized) → Q•- + 2 H+P + Cyt c (reduced)

QH2 + Q•- + 2 H+N + Cyt c (oxidized) → Q + QH2 + 2 H+

P + Cyt c (reduced)

QH2 + 2 H+N + 2 Cyt c (oxidized) → Q + 4 H+

P + 2 Cyt c (reduced)

Why are 2 QH2 required to transfer 2 e- to 2 cytochrome c molecules?

What happens to the “extra” QH2 molecule in step 4 of the Q cycle?

Cytochrome C

Cytochrome c (Cyt c) is responsible for transporting one electron at a time from complex III to complex IV using an iron-containing heme prosthetic group.

Oxidized Cyt c contains ferric iron (Fe3+) in the heme group, reduced Cyt c contains ferrous iron (Fe2+).

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Complex IV: Cytochrome c oxidase

Complex IV accepts electrons one at a time from Cyt c and donates them to oxygen to form water.

In the process, 2 net H+ are pumped across the membrane using a conformational-type mechanism similar to complex I.

In addition, 2 H+ from the matrix side are used to reduce 1/2 O2 to produce 1 H2O.

Combining proton movement across the membrane in complex III and IV, results in 6 net H+

4 H+N

2 H+P

Experimental measurements demonstrate 3 H+ are required to synthesize 1 ATP when they flow back down the electrochemical proton gradient through the ATP synthase complex, and 1 H+ is needed to transport each negatively-charged Pi molecule into the matrix.

ATP Currency Exchange Ratios of NADH and FADH2

ATP Currency Exchange Ratios of NADH and FADH2

Taking into account the requirement of 3 H+/ATP synthesized, and the use of 1 H+ to translocate ADP, the total is 4 H+/ATP.

We can now see where the ATP currency exchange ratios of

~2.5 ATP/NADH and ~1.5 ATP/FADH2 come from:

Oxidation of NADH starting at complex I yields:

10 H+/4 H+ = 2.5 ATP

Oxidation of FADH2 starting at complex II yields:

6 H+/4 H+ = 1.5 ATP for FADH2