Electron Transport System: The Chemiosmotic Theory, redox reactions of the electron transport system, NAD/ATP exchange ratio

Passing the baton is analogous to passing along the e- in the ETS

Rotenone (rat poison) blocks etransport through FeS clusters

Hydrogen cyanide is a deadly gas that blocks e- transport from complex IV to O2 in the ETS

Key Concepts in the Electron Transport System

The Electron Transport System converts redox energy into protonmotive 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.

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 membranebound ATP synthase complex in response to a chemical (H+ concentration) and electrical (separation of charge) differential.

Overview of Chemiosmotic Theory

Electron Transport System

FADH2

Ox Phos ATP synthase complex

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 (psi) Separation of charge is due to build-up of positivelycharged protons (H+) and negative hydroxyl ions (OH-)

Basic Components of the Chemiosmotic Theory

In mitochondria, the contribution of (V) to G is actually greater than that of pH (the pH across the mitochondrial membrane is only 1 pH unit) In chloroplasts, the pH contribution to G is much more significant with pH close to 3 pH units Change in free energy (G) for a membrane transport process is the sum of the ion concentration (RTln(C2/C1)) and the membrane potential (ZFV) In mitochondria, the ZFV term makes a larger contribution than does RTln(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 Mitchells 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.

An Overview
Biological oxidations are catalyzed by intracellular enzymes. The purpose of oxidation is to obtain energy. Electron Transport: Electrons carried by reduced coenzymes (NADH or FADH2) are passed sequentially through a chain of proteins and coenzymes (so called electron transport chain)to O2 . Oxidative Phosphorylation: Coupling eTransport (Oxidation) and ATP synthesis (Phosphorylation) . It all happens in mitochondrion or at the inner mitochondrial membrane (eukaryotic cells)

Fumarate+2H++2e- succinate 0.031 FAD+ 2H++2e- FADH2 0 NAD++ 2H++2e- NADH+H+ -0.32

Succinate+FAD Fumarate+ FADH2 G' = - n F E' Eo' = Eo'(acceptor) - Eo'(donor) G =-2*96485*(0-0.031)= 5.98KJ/mol
Succinate+ NAD+ Fumarate+ NADH+H+ G =-2*96485*(-0.32- 0.031)= 67.7KJ/mol
Removal of H across a C-C bond is not sufficiently exergonic to reduce NAD+,but it does yield enough energy to reduce FAD. Thats why succinate dehydrogenase uses FAD other than NAD+ as coenzyme.

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 oxidoreductase 4. 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 (E') of conjugate redox pairs

Electrons flow spontaneously in this direction

FADH2

The stoichiometry of "proton pumping" is: 4 H+ in complex I 4 H+ in complex III 2 H+ in complex IV (10 H+/NADH and 6 H+/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 (intermembrane 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 reformed 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.

2 H+P

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+P

2 H+N

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+).
mystery protein revealed

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

Sequence of Respiratory Electron Carriers

Inhibitors in green

Electron Transport Chain contd

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------(a) Complex I NADH-CoQ reductase G = -16.6 kcal/mol (ATP)

The Four Electron Transport Complexes in the Inner Mitochondrial Membrane Respiratory Chain

(b) Complex II Succinate CoQ reductase . G = -1.6 kcal/mol (no

ATP)

(c) Complex III CoQ- Cytochrome c reductase, G = -10.1 kcal/mol (ATP)

(d) Complex IV Cytochrome c oxidase G = -19.8 kcal/mol (ATP)

ATP synthesis at F1 results from repetitive comformational changes as rotates

rotates 1/3 turn- energy for ATP release

ATP Currency Exchange Ratios of NADH and FADH2

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

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 10 starting H+/4 at H+ complex = I 2.5 yields: ATP

Oxidation

of

FADH2

starting

at

complex

II

yields:

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

Transport of various metabolites in and out of mitochondria uses electrochemical gradient energy

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

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 Questions
3. What are the key enzymes in the electron transport and oxidative phosphorylation pathway? ATP synthase complex the enzyme responsible for converting protonmotive 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 inhibitors 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.

Control of ATP producing pathway

Alfonse, Biochemistry makes my head hurt!!