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7.3 outline the discovery of ATP synthesis in the mid 20th century in terms of: the discovery of photophosphorylation in chloroplasts of plants the discovery that ATP synthesis involves an electron transfer reaction occurring across a membrane
outline the discovery of ATP synthesis in the mid 20th century in terms of:
- the discovery of photophosphorylation in chloroplasts of plants
- the discovery that ATP synthesis involves an electron transfer reaction occurring across a membrane
- The light independent reaction of photosynthesis requires considerable energy, 2870kJ per mole of 6-carbon sugar formed. It requires 12 NADPH and 18ATP produced by the light dependent reaction. During the light dependent reaction, electrons are transferred and reducing power in the form of NADPH is generated. (see Biochemistry 9.9: 4 The light reaction.)
- Coupled with the electron transfers of the light dependent reaction is the movement of protons (H+, hydrogen ions) from the stroma of the chloroplast, across the thylakoid membrane into the space or lumen inside the thylakoid.
- The thylakoid membrane does not allow the passive diffusion of H+ and so an electrochemical gradient builds up, the inside of the thylakoid becoming positive due to the accumulation of hydrogen ions.
- The movement of the protons across the thylakoid membrane back to the stroma in protein channels drives the synthesis of ATP by the enzyme ATP synthase. The protons cause a conformational (or shape) change in the ATP synthase. This light driven synthesis of ATP is called photophosphorylation.
- In 1961 Peter Mitchell proposed the chemiosmotic hypothesis. He proposed the hypothesis for the mitochondrial membrane and the production of ATP during respiration but the first proof of the hypothesis was for photophosphorylation.
- His hypothesis was based on the following observations:
- ATP synthesis appeared to be membrane based
- An electrochemical gradient based on a H+ concentration gradient was generated when respiration occurred.
- Mitchell proposed that ATP synthesis was driven by an electrochemical gradient across a membrane rather than by the “intermediate” sought by other researchers. He proposed that the gradient was produced by electron transport and that the flow of protons back across the membrane was coupled to ATP synthesis
- In 1966 Jagendorf and Uribe proved that photophosphorylation was chemiosmotic. In the experiment they incubated chloroplasts at pH 4 (hydrogen ion concentration = 1 x 10- 4 molL-1) . They:
- added ADP and radioactively labeled 32P, then quickly raised the pH to 8.
- detected ATP containing radioactive 32P as the difference in pH on the inside and the outside of the chloroplasts became equal.
- carried out the experiments in the dark.
- In 1974 Efraim Racker and Walther Stoeckenius confirmed Mitchell’s chemiosmotic hypothesis using light and bacteriorhodopsin, the equivalent of chlorophyll in the bacteria Halobacterium halobium.
- Work by John Walker and Paul Boyer has determined the structure of ATP synthase. They shared the Nobel Prize for their work in 1997.
- In your research you may have read the terms ”cyclic” and “non-cyclic” when referring to photophosphorylation. Cyclic photophosphorylation is less than 5% of the rate of non-cyclic photophosphorylation, depends only on PS I and does not produce NADPH. The notes provided here, refer to non-cyclic photophosphorylation.
- Mitchell was not able to prove the proton gradient produced ATP when he proposed his hypothesis. Later experiments using change in hydrogen ion concentration (pH) and mitochondria showed that ATP was produced when the H+ crossed the membrane. Peter Mitchell was awarded the Nobel Prize in 1978 for his proposal of what by that time had become the chemiosmotic theory.
- The increased pH is equivalent to a reduced H+ ion concentration on the outside of the chloroplast. The H+ ions move out of the chloroplast along the concentration gradient by diffusion (from more concentrated to less concentrated). This change in pH was meant to be equivalent to the effect of the light dependent reaction on the chloroplasts.