Cells rely on energy carriers, most notably ATP, to perform cellular work. ATP fuels so many reactions that a person may use as many as 1025 of these molecules every day. The process for making ATP during cellular respiration (in mitochondria) and photosynthesis (in chloroplasts) is known as chemiosmosis.
In chemiosmosis, proton (H+) diffusion is coupled to ATP synthesis. When protons build up on one side of a membrane, they form an electrochemical gradient across the membrane. The proton concentration gradient and the electric charge difference constitute a source of potential energy called the proton-motive force. This force tends to drive the protons back across the membrane. Protons diffuse across the membrane through a specific proton channel, called ATP synthase, which couples proton flow with the formation of ATP.
The chemiosmotic mechanism was first proposed by Peter Mitchell in 1961 and has since been widely confirmed through experimentation, beginning with an experiment using chloroplasts.
In 1961, Peter Mitchell proposed a mechanism, called the chemiosmotic mechanism, to describe how mitochondria and chloroplasts produce ATP. He proposed that a gradient of protons forms across membranes in these organelles and that the movement of protons down this gradient, across the membrane, provides the energy to produce ATP. A number of investigators later provided experimental confirmation for Mitchell's ideas.
The concept of pH is important in these experiments. In this example, which side of the membrane would represent pH 7, and which would represent pH 8?
The compartment with a higher concentration of protons has a lower pH. A change of 1 pH unit means a tenfold change in proton concentration.
In one experiment to test the chemiosmotic mechanism, investigators broke open chloroplasts to expose their internal compartments, called thylakoids, that contain the chloroplast's ATP synthase. They intended to use rapid changes in pH around the thylakoids to create a proton gradient. The broken chloroplasts were first preincubated in an acidic medium of pH 3.8. The thylakoids were incubated until equilibrium was reached and the interior of the thylakoids became acidic.
The broken chloroplasts were then moved to a medium with a relatively low proton concentration. The medium also contained ADP and Pi, the building blocks of ATP. These conditions set up a proton gradient across the thylakoid membrane.
The protons moved down the concentration gradient (out of the thylakoids), from high concentration to low, in the process driving the synthesis of ATP. Although chloroplasts normally make ATP during photosynthesis in the light, in these experiments light was not required—just the proton gradient across the thylakoid membrane. This experiment confirmed the chemiosmotic mechanism, in which a proton gradient drives the production of ATP.
A second experiment demonstrated that ATP synthase was indeed the catalyst for the production of ATP driven by the proton gradient. Investigators created artificial vesicles to mimic the membranes involved in chemiosmosis. To these vesicles, they added a light-driven proton pump, called bacteriorhodopsin, from bacteria.
When illuminated, the pH of the external solution increased, meaning that protons were pumped into the vesicles. The proton concentration became higher inside the vesicles than outside, creating a proton gradient.
If ATP synthase from the mitochondria of cow cells is also incorporated into the vesicles, the vesicles formed ATP. The investigators concluded that ATP synthase, acting as a proton channel, is necessary for ATP synthesis.
Which of these scenarios would you predict to result in the formation of ATP?
For the production of ATP, a vesicle requires ATP synthase as a catalyst. Vesicles that have a proton gradient across the membrane already or that have the means of creating the gradient using bacteriorhodopsin and light will be able to drive ATP synthesis.
The chemiosmosis hypothesis was a bold departure from the conventional scientific thinking of the time, which did not consider a role for membranes in the process of ATP synthesis.
The chemiosmotic mechanism involves a complex of transmembrane proteins—including a proton channel and the enzyme ATP synthase—that couples proton diffusion to ATP synthesis. The potential energy of the proton gradient, or the proton-motive force, is harnessed by ATP synthase. ATP synthase acts as a channel allowing protons to diffuse back across the membrane (from high proton concentration to low), and it uses the energy of that diffusion to make ATP from ADP and Pi.