Adenosine triphosphate (ATP) is a coenzyme produced in cellular mitochondria and is not only integral to cellular metabolism but also to life. The mitochondrial production of ATP, which occurs through a complex process called oxidative phosphorylation, has been determined to rely heavily on the enzyme ATP synthase. This determination was the result of a complex experiment that attempted to prove not only that ATP synthase was responsible for production of ATP but also the mechanism of that production as well. ATP synthase is a multisubunit complex with four main parts, each made up of multiple polypeptides (Reece and Campbell 2009).
One of those subunits, F1, has been found to ‘function as a rotary molecular motor’ (Itoh et al. 2004). Also known as F1-ATPase, the F1 unit is attached to the F0 unit, an integral membrane protein with the subunit structure a,b2,c12 – the c subunits forming a circular array with a proton channel formed between the a and c subunits (Meisenberg and Simmons 2006). The ATP synthase motor turns in a clockwise rotation resulting in the hydrolysis of ATP to ADP. In the Itoh et al (‘Itoh’) experiment ATP was shown to be produced as a result of counterclockwise rotation of ATP synthase.
Because ATP synthase turns clockwise in nature, the investigators developed a way to rotate the motor counterclockwise by way of attaching a small magnetic bead to the motor. The F1 subcomplex was attached to a glass surface with histidine residues. The protein Streptavidin, which has a high affinity for biotin, was then used to coat the magnetic bead. The ?-subunit of ATP synthase was then biotinylated so it would bind to the Streptavidin-coated magnetic bead which could now be rotated (counterclockwise) with magnets.
To determine if the counterclockwise rotation of ATP synthase resulted in the production of ATP, the luciferin-luciferase reaction was used. Luciferase catalyzes a reaction between ATP and luciferin which results in a photon emission (light production). The use of luciferin-luciferase meant that if ATP synthase, when rotated counterclockwise, actually produced ATP, light would be emitted. This occurred in the Ihto study and further proved the mechanism of ATP synthesis. As with all experiments, this one was not without its limitations. The otors, although manipulated in the lab, were not synthetic. ATPase motors are found throughout nature in ATP-producing organelles (eg bacteria, plants, fungi) and other motors, similar in structure and function, can also be found in such places as bacterial flagella. The source of the motors in this study derived from the Bacillus strain. Although using a motor derived from a living organism is the most scientifically sound method of studying its function, it also presents a potential problem in that the motors may not function in vitro as they do in vivo.
This potential problem was both realized and compounded by the addition of the magnetic beads. Specifically, the motors became saturated and their rotary speed limited. Although it proved challenging, it was not insurmountable. After a number of setbacks and continuous ‘fine tuning’ of the motor, the researchers were able to conduct multiple rounds of the experiment and accurately measure their results (with luminescence from the luciferin-luciferase reaction) to conclusively demonstrate the function of ATP synthase.