4.4.4—
Phosphorylation and Thylakoid Structure

Three main hypotheses have been proposed for the mechanism of ATP formation in mitochondria and chloroplasts (Slater, 1971), but here we will briefly consider only one of these, the Mitchell chemiosmotic hypothesis (Mitchell, 1966; see also chapter 5). The fundamental concept of the chemiosmotic hypothesis is that of vectorial flow of protons across the mitochondrial inner membrane or the thylakoid membrane. Mitchell proposed that the electron transport chains of mitochondria and chloroplasts consist of alternate electron and hydrogen carriers, organized in a vectorial fashion across the membrane. Transport of electrons through the chain of carriers results in the net transfer of protons across the membrane. For the thylakoid membrane, protons are transported from the outside (stroma or matrix) to the inside intrathylakoid space. This is depicted in Fig. 4.5 (Trebst, 1974). In this scheme, reduction of plastoquinone by Q

Figure 4.5
Photosynthetic electron flow, showing vectorial arrangement of carriers and the
movement of protons from outside to inside of the thylakoid. (From Trebst, 1974.)

occurs on the outside of the thylakoid and oxidation by cytochrome ¦ on the inside, resulting in the transfer of 2 protons for each 2 electrons transported from water to NADP⁺ . Water oxidation is considered to take place on the inside to produce 2 protons per 1/2O₂ . According to the Mitchell hypothesis, the protons move back through the membrane through special channels which contain the coupling factor or ATPase, and result in formation of ATP from ADP and inorganic phosphate. As already discussed, part of the ATPase (CF₁ ) is on the outside of the thylakoid membrane, and part (CF₀ ) is embedded in the membrane. It is not yet established whether the proton gradient across the thylakoid membrane is an essential intermediate in the process of ATP formation during photosynthetic electron flow. However, with thylakoid membranes it has been shown

that ATP formation can be driven by a pH gradient created artificially (Jagendorf & Uribe, 1966). Chloroplasts are suspended in a buffer of low pH (pH < 5) and then transferred to a buffer of pH 8, containing ADP and inorganic phosphate.

According to the Mitchell hypothesis, the energy-conserving sites are identified with the proton-releasing sites on the inside of the thylakoid. Substances such as ammonia which uncouple ATP formation from electron transport in the thylakoid membrane (termed uncouplers) are considered to destroy the proton gradient by transporting the protons across the membrane.