Formation of ATP by Photophosphorylation | Bioenergetics

In this article we will discuss about the process of formation of ATP by photophosphorylation.

Photosynthesis: Light Phase and Dark Phase:

Heterotrophic organisms collect from the external medium, comparatively simple organic molecules which can serve as carbon-containing skeletons for their syntheses or as energy source (ATP) when they are oxidized during mitochondrial reactions of respiration according to the general reaction:

C₆H₁₂O₆ + 6O₂ → 6CO₂ + 6H₂O + energy (ATP)

These organic molecules are ultimately synthesized in their very large majority by photosynthetic autotrophic organisms. Higher plants and most algae do have structures, chloroplasts and an enzymatic equipment which enables them to utilize light energy to perform the synthesis of simple sugars from CO₂ and H₂O according to the general photosynthetic reaction:

6CO_z + 6H₂O + light energy → C₆H₁₂O₆ + 6O₂

Experiments with water containing the isotope ¹⁸O showed that the oxygen released originates from the cleavage of water. It therefore appears that, in higher plants, photosynthesis reactions utilize light energy to extract hydrogen atoms from water while releasing oxygen and transfer them to CO₂ in order to form reduced carbon-containing products.

This shows the difference with respiration where on the contrary, various reduced substrates are oxidized into CO₂, yielding hydrogen atoms which combine with oxygen to form water while energy (ATP) is liberated.

The photosynthetic reduction of CO₂ involves a complex system of reactions which can be grouped in two principal phases:

1. The light phase in which light energy absorbed by chlorophyll is converted into chemical energy usable by the cell and during which ATP and NADPH are formed;

2. The dark phase in which, thanks to ATP and NADPH thus formed, CO₂ is reduced into simple sugars during a cycle of enzymatic reactions called Calvin cycle. For a proper understanding of these reactions one would need a good knowledge of the metabolism of carbohydrates, which is the reason why we are studying them (see fig. 4-45).

After being synthesized in the chloroplast, these simple sugars are exported to the cytoplasm where they will play a role equivalent to that of organic molecules collected from the external medium by heterotrophic organisms.

They will enter the cellular metabolism and will be transformed through anabolic reaction chains into diverse molecules. Those molecules which will be degraded through catabolic reaction chains, will be oxidized into CO₂ and H₂O in the mitochondria by the mechanisms of respiration, with formation of the ATP required for the various cellular activities.

In plant cells, chloroplasts and mitochondria therefore co-exist and bring about simultaneously photosynthetic reactions which consume light energy and respiratory reactions of degradation which liberate energy usable by the cell (ATP), in varying proportions depending on external conditions (light, temperature, humidity etc.).

Chloroplasts and Chlorophyll:

All the photosynthesis reactions in higher plants and eucaryotic algae take place in chloroplasts, organelles which present several structural and functional similarities with the mitochondria, both fulfilling the energetic functions of the cell. The number of chloroplasts per cell varies from one (in some algae) to about ten (in higher plants) and their size is around 10 micrometers.

Like the mitochondria, they are surrounded by an envelope consisting of two membranes: the outer membrane, very permeable to ions and small molecules and the inner membrane, impermeable and in which numerous transport proteins perform the exchanges with the cytoplasm.

The chloroplastic com­partment thus defined contains the stroma, which is similar to the matrix of mitochondria. It consists of a non-structured mixture of soluble moleculcs including the enzymes of the Calvin cycle, several copies of the chloroplastic chromosome as well as the systems of replication, transcription and translation of this genetic information.

The chloroplastic space also contains an important system of membranes, similar to the crests of mitochondria. These membranes, called thylakoids, very rich in galactolipids form flattened, inter­connected vesicles arranged in a stack to form the grana. The primary steps of photosynthesis corresponding to light reactions take place in thylakoids.

They contain numerous multi-molecular assemblies consisting of proteins, chlorophyll, cytochromes, quinones… (see fig. 3-15) which bring about the reactions of light energy absorption, photochemical conversion and electron transport with formation of ATP and NADPH.

Chlorophylls are the pigments characteristic of all photosynthetic organisms. Chlorophyll a and chlorophyll b, present in all chloroplasts of higher plants and numerous algae are very similar in structure (see fig. 3-13). Both have a tetrapyrrole ring similar to that of the heme (see fig. 1-29) but with a magnesium atom.

A long hydrocarbon phytyl side chain confers on these molecules a liposoluble character which enables them to establish hydrophobic bonds with some of the intrinsic proteins of thylakoids to form chlorophylls-proteins com­plexes. However, chlorophyll b has a formyl group on carbon β of one of the pyrrole rings in place of a methyl group present on chlorophyll a.

The colour of chlorophylls is due to their structure which enables them to absorb light at some wavelengths as per the mechanisms described in the following paragraph. The two peaks near 430 nm and 680 nm visible on the absorption spectrum (solid line in figure 3-14) show that chlorophyll a absorbs light in the violet-blue and red regions respectively. Its green colour therefore corresponds to the portion of the spectrum which has not been absorbed and is therefore transmitted or reflected.

The absorption spectrum of chlorophyll a coincides fairly well with the action spectrum of photosynthesis, i.e. with the curve which represents the efficiency of photosynthesis as a function of wavelengths (dotted line in figure 3-14). Chlorophyll a, therefore appears, at least in higher plants and algae, as the major pigment responsible for the photosynthetic reactions.

However, other pigments participate, together with chlorophyll a, in the process of collection of photons: chlorophyll b in higher plants and green algae, chlorophyll c and carotenoids in brown algae, phycocyanines and phycoerythrines in blue and red algae. These accessory pigments which are, like chlorophylls, associated with proteins of thylakoids permit the collection of photons in regions of the spectrum not absorbed by chlorophyll a.

Absorption of Light by Chlorophyll:

The characteristics of light and matter are such that interactions may take place between the two.

Light corresponds to the very limited portion of the spectrum of electromag­netic radiations, between 400 nm (blue) and 700 nm (red), to which our eye is sensitive. The fringe areas correspond to the ultraviolet (< 400 nm) and the infrared (>700 nm).

Light consists of a flux of photons which are particles charged with a defined quantity of energy called energy quantum. The energy of photons is inversely proportional to their wavelength and therefore decreases from the violet end to the red end of the spectrum.

Matter consists of nuclei surrounded by electrons placed on orbitals, of energy determined by their distance to the nucleus. These electrons can be shifted by the energy of an absorbed photon. Thus, when a light beam strikes a selenium surface, the energy of photons can extract from the latter a number of electrons which increases as the intensity of light increases (principle of the photoelectric cell).

In the case of coloured molecules and chlorophyll in particular, the energy of an absorbed photon can shift electron from its initial orbital up to an orbital of higher energy, more distant from the nucleus. The energy of the absorbed photon is therefore transformed into energy of electronic excitation.

However, only those photons will be absorbed whose energy is equal to the difference of energy between the two orbitals; the others, corresponding to the non-absorbed wavelengths will be reflected or transmitted, conferring their characteristic colour on the molecules thus exposed to white light.

In the case of selenium atoms, the energy of photons is sufficient to pull out of the attraction of the nucleus, electrons which can then create an electric current. In the case of coloured molecules, the excited state of an electron which has changed its orbital under the impulse of a photon is very unstable.

In most cases, the displaced electron falls back on its original orbital (return to the fundamental state) liberating the stored energy in the form of heat or fluorescence (emission of photons).

In some cases, this excited state can be transferred by resonance to a neighbouring identical molecule which is then excited in its turn. Sometimes, the excited electron can be shifted to another molecule of different nature; this reac­tion which thus produces an oxidized molecule (loss of electron) and a reduced molecule (gain of electron) is a photochemical reaction.

Reaction Centers and Antenna Complexes:

The excited state of a chlorophyll molecule, created by the absorption of a photon, is therefore very unstable. In vitro, the displaced electron falls back on its starting orbital, liberating the potential energy in the form of heat or fluorescence. In vivo, this electron can on the contrary, be transferred to a neighbouring molecule and produce in it a reducing power utilizable by the cell.

This photochemical reaction takes place in the reaction centers where a primary electron donor chlorophyll molecule a is closely associated with a primary electron acceptor molecule. A series of oxidation-reduction reactions involving chains of electron carriers very similar to those involved in mitochondrial respiratory chains then permit the flow of this electron towards a terminal acceptor which is then in a reduced form.

But the rate of absorption of the photon by a chlorophyll molecule, even in bright sunshine, is too small to produce an appreciable flow of electrons. On the other hand, detailed studies have shown that a very small proportion of chlorophylls (about 1/200) is involved in the photochemical conversion.

The great majority of chlorophylls are organized in antenna complexes comprising each about 200 molecules of chlorophyll and each associated with a reaction center. The unique function of these antenna complexes is to absorb light energy; transfers by resonance between the chlorophyll molecules then channel the excitation energy towards the reaction centers whose photochemical activity is multiplied by about 200.

These energy transfer and photochemical conversion reactions are very rapid (a few nanoseconds) and require a very accurate structural organisation. This essential condition is fulfilled by the association of the molecules involved (chlorophylls, primary acceptors…) with various proteins which are themselves organized in stable multi-molecular structures (see fig. 3-16).

5. Electron Transport and Photosynthetic Phosphorylation:

We have seen in the foregoing, that in the plant cell, the “excited” electrons can leave the chlorophyll molecules activated by light to be taken over by an electron carrier system. In fact, it appears that the electrons removed from chlorophyll a of the reactive center can take two different pathways; one leading to the synthesis of ATP and the other to the formation of ATP and NADPH.

We will study these two possibilities and see how — in each case — the problem of the regeneration of chlorophyll a can be resolved. All the mechanisms of photosynthesis are not yet known accurately; we are presenting here a simplified diagram taking into consideration the processes which are apparently the most probable at the present time.

We know that in green plants, chlorophyll is involved in two photosensitive systems playing a role in two different photochemical reactions: photosystem I (PS I) and photosystem II (PS II).

A. Non-Cyclic Photophosphorylations with Formation of ATP and NADPH:

In this principal pathway, the electrons removed from the chlorophyll of the reactive center are driven by a chain of carriers towards a terminal acceptor NADP⁺ which is reduced to NADPH. The chlorophyll of the reactive center returns to the fundamental state by collecting an electron from an ultimate donor AH which is therefore oxidized

AH → A + e^– + H⁺

But in the case of higher plants, where this donor is water, the energy of two photons, transformed by two successive photochemical reactions located in the two photosystems, is necessary for the displacement of an electron up to NADP⁺.

In photosystem II, the light energy absorbed by the chlorophylls of the collector antenna (chlorophylls a and b) is transferred to the chlorophyll of the reactive centers (P 680) where a first photochemical reaction takes place and allows the shift of an electron to a primary acceptor (a pheophytin) and then to the quinones QA and QB.

The consecutive oxidation of water liberates an electron which allows the regeneration of the chlorophyll of the reactive center via an intermediate carrier containing manganese (see fig. 3- 15). These various elements are associated with different proteins organised in a first type of multi- molecular complex (see fig. 3-16).

The electrons shifted to QB are then carried by a set of free plastoquinones (PQ) in the lipidic phase of thylakoids to a second type of membrane protein complex containing the cytochromes b₆, f and an iron-sul­phur protein. The energy liberated during this transport establishes a trans­membrane gradient of protons which will be utilized for the synthesis of ATP (see paragraph VI) by another protein complex forming ATP synthetase (see fig. 3-16).

In photosystem I, the light energy absorbed by the antenna (mostly chlorophyll a) allows, in the course of a second photochemical reaction, the displacement of an electron from the chlorophyll of the reactive center (P 700) to a primary acceptor X of unknown nature, then to another iron-sulphur protein. These compounds are organised in a fourth type of membrane protein complex (see fig. 3-16).

The electrons are then transported up to NADP⁺ via ferredoxin (Fd) and ferredoxin NADP reductase (FNR). The regeneration of the chlorophyll of the reactive center (see fig. 3-15) is accomplished with the electron originating from photosystem II, starting from cytochrome f via plastocyanin (PC). In the overall balance, the energy of the photons absorbed, transformed by these two photochemical reactions in series, allows the forma­tion of the reducing power (NADPH) from electrons removed from water and the synthesis of ATP.

B. Cyclic Phosphorylations and Formation of ATP Only:

The importance of these reactions is not yet fully understood; they involve only photosystem 1. Their juxtaposition to the non-cyclic phosphorylations, leading to a formation of ATP alone, probably adjusts the ATP/NADPH ratio to the requirements of the chloroplast. The electrons displaced by the photochemical reactions are, as in the previous case, carried up to ferredoxin.

But instead of being directed towards NADP⁺, they fall back on cytochrome b₆ of the complex cytochrome f, b₆, iron-sulphur protein. With the participation of plastoquinones, they establish a transmembrane gradient of protons utilised for the synthesis of ATP.

The electrons then join, via the plastocyanins, the chlorophyll P 700 of the reactive center which is thus regenerated. In this case, the energy of the photons absorbed leads only to a cyclic flow of electrons allowing only the synthesis of ATP.

In conclusion, respiration as well as photosynthesis, involve movements of electrons in specialised membranes. In mitochondria these electrons originate from various organic substrates subjected to the action of the corresponding dehydrogenases whereas in chloroplasts, they are removed from the chlorophyll by the energy of light, in mitochondria, the electrons finally reach the oxygen, the reduction of which forms water; in chloroplasts, the electrons can either allow the reduction of NADP⁺, or return to the chlorophyll and allow the restoration of its fundamental state.

As in mitochondria, the flow of electrons along the chain of carriers makes it possible to store a part of the energy in the form of a transmembrane gradient of protons utilized for the formation of ATP. Only the source of this energy differs; in mitochondria, it is the chemical energy of various organic substrates, while in the chloroplasts, it is the light energy of the absorbed photons.

Finally, it must be indicated that photosynthetic organisms (green plants, microorganisms etc.) possess relay systems of respiratory chains to form ATP through oxidative phosphorylations in the absence of light. Plants thus have mitochondria in addition to chloroplasts.

C. Bacteriorhodopsin and Photophosphorylation:

Chlorophylls are not the only photosensitive molecules which can collect the energy of photons and utilize it to form reductants and ATP for the purpose of syntheses. Some bacteria carry out their photosynthesis thanks to the presence of bacteriorhodopsin, a protein linked to retinal (the mechanism of purple membranes of Halobacterium halobium).