Chemiosmotic coupling

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Coupling the endergonic reaction X → Y to the electrically-driven proton flow:
X + H + (outside) → Y + H + (lbs)

Exergonic reactions provide the energy for the proton flux against the electric field.

As Chemiosmosis or Chemiosmose refers to a mechanism, coupled with the transport processes of biomembranes with central chemical metabolic processes.

By far the most important reaction for the regeneration of the energy carrier ATP from ADP + P i is based on a chemiosmotic process

This endergonic reaction is catalyzed by the enzyme ATP synthase , which is embedded in an electrically charged biomembrane . The electrochemical force of the ion gradient that exists between the inside and outside of the membrane is used by the enzyme to build up the high-energy tri-phosphate bond of the ATP (see Fig. 1 ).

A number of other biological processes are based on this force. So many exergonic reactions chemiosmotisch coupled to the structure of the electric field of the biomembrane (see. Fig. 2 ).

Development of the chemiosmotic theory and the terms used

Overall process of electron transport phosphorylation

Without ATP, every known metabolism breaks down and the organism dies. On the other hand, ATP cannot be stored in significant quantities and has to be constantly regenerated from ADP in the cells. In the human body, each ATP molecule is recovered from ADP and used up approx. 3000 times per day. It has been known since the middle of the 20th century that the main mass of ATP in humans is regenerated through the oxidation of NADH . For years the search for a substance had been unsuccessful which, as an energy-rich intermediate product, brought about the coupling of NADH oxidation with ATP formation.

In 1961, Peter D. Mitchell proposed a chemiosmotic model (also known as the Mitchell Hypothesis ). He denied that NADH oxidation is linked to ATP synthesis through an intermediate. His hypothesis:

  • An electrochemical potential on the mitochondrial membrane is responsible for the coupling .
  • The space outside this membrane has a higher concentration of H + ions than the matrix inside. Hence the name proton gradient comes from .
  • The NADH oxidation takes place in an electron transport chain that functions as an outwardly directed “proton pump” . This proton translocation produces a
  • Proton Motive Force ΔP (Abbreviation PMF , German: Proton Moving Force). The energy of the returning protons provides the energy for the process catalyzed by the ATP synthase.
  • Electron transport phosphorylation is the name for the overall process in which an electron transport chain supplies the energy for the phosphorylation of ADP to ATP.

In 1978 Mitchell was awarded the Nobel Prize in Chemistry for discovering chemiosmotic coupling . Years later, there was still a clear skepticism about the " Mitchell hypothesis ", even in textbooks. It is now undisputed that the chemiosmotic coupling not only plays a central role in the metabolism of animals and plants (in the mitochondria and plant chloroplasts ), but also in almost all microorganisms .

In the older literature still called the chemiosmotic hypothesis , the “ general chemiosmotic theory ” is now generally accepted.

Building and maintaining the membrane potential

Building up and maintaining the membrane potential is based on the export of cations to the outside of the biomembrane. The export requires energy, since the ions have to be transported against a charge and concentration gradient.

The energy of the membrane potential is vital for the cell. If its electrical voltage and the differences in concentration of the ions between the inside and outside are not sufficiently regenerated, the ATP supply to the cell breaks down and it "starves". This also happens when the structural integrity of the membrane is damaged. Every hole leads to a life-threatening "short circuit". The membranes of living cells are always closed compartments.

Almost all organisms use the export of H + . This export is known as proton translocation and occurs through so-called proton pumps.

In biology, pH gradients occur on the membranes of cells and cell organelles ( mitochondria and chloroplasts ). pH gradients on membranes are essential for the energy metabolism of all known organisms. These gradients are not static, but are in a quasi- steady flow equilibrium . Protons are actively pumped against the concentration gradient ( proton pump ), while at the same time the potential of the gradient is used energetically. The overall process is sometimes called the proton flow.

The energy to build up the membrane potential comes either from the difference in the redox potentials during the oxidation of high-energy substances ( oxidative phosphorylation ) or from the light energy ( photophosphorylation ).

Compartments and energy source of membrane potentials of different organisms
Organism or organelle Outside Inside Energy source
Archaea Extracellular space Cytoplasm Redox potential / light
bacteria Periplasm Cytoplasm Redox potential / light
Chloroplasts in Plants Thylakoid interior Stroma Redox potential / light
Mitochondria in eukaryotes Intermembrane space matrix Redox potential

Oxidative phosphorylation

Mechanism of energy production by the hydrogen-oxidizing bacterium Aquifex aeolicus

In the respiratory chain of the mitochondria , several protein complexes can function as proton transporters: In addition to NADH dehydrogenase (complex I), cytochrome c reductase (complex III) (via the Q cycle ), and cytochrome c oxidase ( Complex IV). The energy comes from the high redox potential difference between NADH or FADH 2 and oxygen. The gradient is strengthened by the consumption of protons during the formation of water from oxygen and protons by the cytochrome c oxidase.

In chemotrophic prokaryotes a plurality of electron transport chains has been found to act as proton pump. The ATP generation in chemo- litho -trophen organisms is usually due to the fact that the electron transport chain extending from an outer oxidized fuel to an internal reduced oxidizing agent.

Oxidative phosphorylation is also widespread in anaerobic , chemotrophic organisms ( anaerobic "respiration" ). Many of these organisms use an alternative metabolic pathway when there is a lack of oxygen, namely substrate chain phosphorylation . It is not based on the chemiosmotic mechanism and takes place in the cytoplasm.

Photophosphorylation

Chlorophil and electron transport chain

Phototrophic bacteria and some archaea also regenerate ATP through electron transport chains in which membrane proteins act as proton pumps. In sulfur-free purple bacteria , a cytochrome '' bc '' 1 fulfills this function under anaerobic conditions . The energy supplier here is light, which drives a cyclic electron transport via a light-collecting complex.

In the oxygenic photosynthesis of green plants and cyanobacteria , the cytochrome b 6 f complex (Cyt b 6 f ) of the redox chain functions as a proton pump.

  • The energy comes initially from the redox potential of the electrons, which  are transported through the redox chain from photosystem II (PS II) to photosystem I (PS I) during acyclic ATP formation . The energy of the electrons comes from the excitation by the light in photosystem II.
  • In cyclic phosphorylation, electrons in photosystem I are excited by light and transferred back to cytochrome b 6 via ferredoxin .

The proton gradient is reinforced by the fact that the water is split into oxygen and protons on the side of the thylakoid interior, i.e. the proton concentration is additionally increased. On the stroma side, NADPH is formed with the consumption of protons.

Proton pump rhodopsin

Finally, Haloarchaea can use light energy directly through bacteriorhodopsin to build up a membrane potential. In contrast to all the mechanisms described so far, this very simple mechanism is not based on a redox chain.

ATP synthase as a proton pump

The enzyme ATP synthase is found even in anaerobic organisms that do not obtain ATP according to the chemiosmotic mechanism, but through substrate chain phosphorylation . In these organisms, however, it serves as a proton pump to maintain the pH value required inside the cell. In addition, these living beings also use the energy of their membrane potential for numerous processes.

Energizing membranes

Membrane composed of the phospholipid lecithin, which is energized by a proton gradient.

The double lipid layer of biomembranes is due to its chemical structure - inside there are hydrophobic (water-insoluble and water-repellent) molecule chains - impermeable to water and ions such as H 3 O + . As a result, it forms a barrier for the spontaneous compensation of a pH difference between the two sides of the membrane.

The hydrophilic “heads” of the molecules are located on the outside of the double lipid layer. Examples of membrane lipids are glycolipids and phospholipids . The heads of the phospholipids in particular are amphoteric , so they can absorb positive or negative charges.

These groups of molecules act as buffers against changes in the pH value . Negatively charged phosphate groups can be “protonated” at low pH values ​​and are no longer negatively charged as phosphoric acid residues. In the alkaline range, -NH 3 + groups can be converted into neutral -NH 2 amino groups.

If there is a difference in pH, there are different electrical charges on both sides of the membrane, positive in an acidic environment and vice versa. Biomembranes can therefore store electrical energy in the form of membrane potential like a capacitor .

Voltage and energy content of a proton gradient

The force of the proton gradient ΔP on a biological membrane is made up of two components, one chemical and one electrical. The connection between proton gradients and energy production in a cell is described by the chemiosmotic theory of Peter D. Mitchell (1961). The use of proton gradients on biomembranes may have arisen from the use of geological proton gradients, e.g. B. Black smokers .

  1. The difference in the concentration of H + ions ΔpH .
  2. The tension on the membrane, membrane potential ΔΨ :

ΔP = ΔΨ - Z × ΔpH
( Z = 2.3 R T / F. The latter formula roughly states: proton gradient and the membrane charge add up depending on the temperature, because otherwise they only have one constant, namely the quotiont of the universal gas constant and Faraday- Constant .)

The relationship between the proton gradient ΔP and the electrochemical potential difference with respect to the protons Δ H + is:

ΔP × F = −Δ H +

Example:

  • When E. coli bacteria grow at a pH value of 6 and have a pH value of 7.8 inside the cell, an electrical voltage of −95 mV is measured on their membrane . According to the latter equation, their proton gradient, which can be used for biological processes, then has a total of −200 mV . This value is enormous, especially since the insulating membrane is only about 2 nm thick.

Use of the membrane potential

Proton gradient and ATP synthesis.

By far the most important use of the membrane potential takes place, as described above, in the regeneration of ATP. There are also a number of other processes that use the pH gradient.

Figure 2 . In the picture on the left, a small part of the cytochrome c formed from nitriteenters the transmembrane complex II. It contains an electron transport chain, the components ofwhich are reducedby inflowing H + to such an extent that NAD + can be reduced to NADHin the cytoplasm.

Reverse electron transport in chemolithotrophic prokaryotes

Chemolithotrophic organisms have to regenerate NADH with weak reducing agents. It has long been debated whether ATP has to be used for this endergonic reaction. In fact, it has been shown that the membrane potential can be used directly for this “reverse electron transport”.

Proton gradient, decoupler and thermogenesis

Newborns and hibernating mammals can gain heat directly from the proton gradient in the brown adipose tissue . To do this, they use so-called decouplers, which enable the (regulated) influx of H + ions into the interior of the mitochondria without generating ATP from ADP. This bypass of ATP synthesis allow UCP proteins ( u n c oupling p roteins) as Thermogenin . They decouple the respiratory chain from ATP synthesis, hence the name decoupler.

In biochemistry decoupler have been known for a long time. When the respiratory chain was explored, z. B. 2,4-Dinitrophenol (DNP) are used. Like other chemical decouplers, DNP's function is based on the breakdown of the pH gradient. The DNP molecules dissolve in the lipophilic interior of the membrane. They give off H + on the inside of the membrane , diffuse to the outside and take up H + again there .

Proton gradient and flagella movement

Bacterial locomotion by flagella is also based - as with ATP synthase - on a proton-driven rotary movement. As with ATP synthase, the engine does not work on the principle of a turbine. Rather, the flow of protons creates a force between the rotating rotor and proteins that are firmly anchored in the cell wall and function as a stator. In contrast to ATP synthase, the molecular mechanism of the scourge motor has not yet been clarified in detail.

The flagella are driven by a basal body that is anchored in the cell wall. In addition to the actual motor, it also includes ring-shaped proteins that function as a pivot bearing. The whip itself is moved passively, similar to a mixer.

The efficiency of this machine is remarkable. The proton-driven, natural “nanomotor” reaches 100,000 revolutions per minute in some organisms.

Alkaliphilic organisms

Fig. Electron and proton transport through cytochromes in Pseudomonas alcaliphila , an alkaliphilic bacterium.

Alkaliphilic bacteria can grow in media that are significantly more basic than their cell plasma. Contrary to earlier assumptions use especially aerobic organisms alkaliphilic H + -driven ATP synthases and H + -translozierende redox chains. Organisms need special mechanisms to build up a membrane potential, since protons released into the alkaline medium would be immediately neutralized. In many of these organisms, negatively charged molecules are increasingly stored in the cell wall, which means that they represent a certain barrier against OH - ions.

Further adaptations are shown schematically in the figure opposite using the example of the gram-negative γ-proteobacterium Pseudomonas alcaliphila .

The organism has two things in common with other alkaliphilic Pseudomonas species that are not found in neutrophilic pseudomonads .

  • When growing in strongly alkaline nutrient media, there are more special c-cytochromes in the periplasm . These not only act as electron carriers from cytochrome c reductase (in the figure coenzyme Q cytochrome c oxidoreductase) to cytochrome c oxidase . Rather, at high pH values ​​they serve as “condensers” and carriers for protons that are released by the respiratory chain on the outside of the membrane. The cytochromes keep the H + cations away from the alkaline medium and transfer the protons to the ATP synthase. Such c-cytochromes have also been found in some gram-positive alkaliphilic bacteria.
  • Even more common in alkaliphilic bacteria is a mechanism with which the organisms can keep the pH value in the plasma in the neutral range they require. As a rule, they have an H + / Na + antiporter (on the left in the picture), which exchanges outwardly translocated H + for sodium cations. The electrical voltage of the membrane is maintained. The electrochemical Na + gradient on the cell membrane is used by akaliphilic organisms for a number of processes that are based on a proton gradient in other organisms.

Active transport processes

Detoxification of a penetrated poison by Antiport

The energy of the membrane potential can also be used for active transport processes. If uncharged low-molecular poisons have penetrated the membrane, they can be partially oxidized and then actively transported out of the cell.

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