Mitochondrial ATP synthase
- Mitochondrial ATP synthase is an F-type ATPase similar in structure and mechanism to the ATP synthases of chloroplasts and eubacteria.
- This large enzyme complex of the inner mitochondrial membrane catalyzes the formation of ATP from ADP and Pi, accompanied by the flow of protons from the P to the N side of the membrane
- ATP synthase, also called Complex V, has two distinct components:
- F1, a peripheral membrane protein, and
- Fo (o denoting oligomycin-sensitive), which is integral to the membrane.
- F1, was identified and purified by Efraim Racker and his colleagues in the early 1960s.
- F1-depleted vesicles cannot make ATP.
- Isolated F1 catalyzes ATP hydrolysis (the reversal of synthesis) and was therefore originally called F1ATPase
- When purified F1 is added back to the depleted vesicles, it re-associates with Fo, plugging its proton pore and restoring the membrane’s capacity to couple electron transfer and ATP synthesis.
- The crystallographic determination of the F1 structure by John E. Walker and colleagues revealed structural details
- Mitochondrial F1 has nine subunits of five different types, with the composition α3ß3γδε.
- Each of the three ß subunits has one catalytic site for ATP synthesis.
- The knob like portion of F1 is a flattened sphere, 8 nm high and 10 nm across, consisting of alternating α and ß subunits arranged like the sections of an orange.
- The polypeptides that makeup the stalk in the F1 crystal structure are asymmetrically arranged, with one domain of the single γ subunit making up a central shaft that passes through F1, and another domain of γ associated primarily with one of the three subunits, designated β-empty
- The structures of the δ and ε subunits are not revealed in these crystallographic studies.
- The Fo complex making up the proton pore is composed of three subunits, a, b, and c, in the proportion ab2c10–12.
- Subunit c is a small (Mr 8,000), very hydrophobic polypeptide, consisting almost entirely of two trans-membrane helices, with a small loop extending from the matrix side of the membrane.
- The ε and γ and subunits of F1 form a leg-and-foot that projects from the bottom (membrane) side of F1 and stands firmly on the ring of c subunits
Rotational Catalysis : Mechanism of ATP synthesis
- Paul Boyer proposed a rotational catalysis mechanism in which the three active sites of F1 take turns catalyzing ATP synthesis
- Three ß subunits assume 3 different conformations
A given subunit starts in the ß-ADP conformation, which binds ADP and Pi from the surrounding medium.
- The subunit now changes conformation, assuming the ß-ATP form that tightly binds and stabilizes ATP, bringing about the ready equilibration of ADP+Pi with ATP on the enzyme surface.
- Finally, the subunit changes to the ß-empty conformation, which has very low affinity for ATP, and the newly synthesized ATP leaves the enzyme surface.
- Another round of catalysis begins when this subunit again assumes the ß-ADP form and binds ADP and Pi
- The conformational changes central to this mechanism are driven by the passage of protons through the Fo portion of ATP synthase.
- The streaming of protons through the Fo “pore” causes the cylinder of c subunits and the attached γ subunit to rotate about the long axis of γ, which is perpendicular to the plane of the membrane.
- The subunit passes through the center of the α3ß3 spheroid, which is held stationary relative to the membrane surface by the b2 and δ subunits
- With each rotation of 120°, γ comes into contact with a different ß subunit, and the contact forces that ß subunit into the ß-empty conformation.
- The three subunits interact in such a way that when one assumes the ß-empty conformation, its neighbor to one side must assume the ß-ADP form, and the other neighbor the ß-ATP form.
- Thus one complete rotation of the γ subunit causes each ß subunit to cycle through all three of its possible conformations, and for each rotation, three ATP are synthesized and released from the enzyme surface.
- One strong prediction of this binding-change model is that the γ subunit should rotate in one direction when FoF1 is synthesizing ATP and in the opposite direction when the enzyme is hydrolyzing ATP.
- This prediction was confirmed in elegant experiments in the laboratories of Masasuke Yoshida and Kazuhiko Kinosita, Jr.
- The rotation of γ in a single F1 molecule was observed microscopically by attaching a long, thin, fluorescent actin polymer to γ and watching it move relative to α3ß3 immobilized on a microscope slide, as ATP was hydrolyzed.
- When the entire FoF1 complex (not just F1) was used in a similar experiment, the entire ring of c subunits rotated with γ.
- The “shaft” rotated in the predicted direction through 360°.
- The rotation was not smooth, but occurred in three discrete steps of 120°.
As calculated from the known rate of ATP hydrolysis by one F1 molecule and from the frictional drag on the long actin polymer, the efficiency of this mechanism in converting chemical energy into motion is close to 100%.