7.4: Oxidative Phosphorylation in Respiration

Oxidative phosphorylation is the mechanism by which ATP captures the free energy in the mitochondrial proton gradient. Most of the ATP made in aerobic organisms is made by oxidative phosphorylation, and not by substrate-level phosphorylation (the mechanism of ATP synthesis in reactions of glycolysis or the Krebs cycle). Some aerobic biochemistry may have evolved in response to the toxic effects of rising environmental oxygen levels. Later elaboration of respiratory metabolism was undoubtedly selected because it turned out to be significantly more efficient at making ATP than were the anaerobic fermentations (such as “complete” glycolysis). In other words, oxidative phosphorylation is more efficient than substrate-level phosphorylation.

CHALLENGE

What abundant environmental substrate of oxidative phosphorylation contributes to its high efficiency?

To summarize, the movement of electrons down the electron-transport chain fuels the three proton pumps that establish a proton gradient across the cristal membrane, which stores free energy. Oxidative phosphorylation then allows the controlled diffusion of protons back into the mitochondrial matrix through the cristal membrane’s ATP synthases, fueling ATP production. We say that the proton gradient has a proton motive force. However, we also recognize that the proton gradient is also a pH gradient, and an electrical gradient (i.e., the difference in electric potential).

The use of this proton motive force to make ATP is regulated. Conditions in the cell control when the energy stored in this gradient will be released to make ATP. The switch that allows protons to flow across the cristal membrane to relieve the proton gradient is a mitochondrial cristal membrane ATP synthase, a tiny, complex enzymatic protein motor.

When this “switch” is open, protons pumped out of the mitochondrial matrix during electron transport can flow back into the matrix through the ATP synthase. For a clear discussion of this complex enzyme, see P. D. Boyer (1997) The ATP synthase – a splendid molecular machine. Ann. Rev. Biochem. 66:717-749.

The splendid molecular machine was discovered when isolated mitochondria were themselves fractionated. When a membrane fraction (Fraction 1) of the organelle was shown to hydrolyze ATP, the putative enzyme responsible was designated an F1 ATPase. Intact mitochondria cannot hydrolyze ATP! In further experiments, vesicles formed from isolated mitochondrial membranes were exposed to high pH in the presence of ADP and inorganic phosphate, resulting in ATP synthesis. Hence, F1 ATPase was redesignated the mitochondrial ATP synthase (or F1 ATP synthase). The capture of free energy of protons flowing through this lollipop-shaped ATP synthase by facilitated diffusion is shown in Figure 7.2.

Figure 7.2: The passive diffusion of protons through the cristal membrane ATP synthase relieves the proton gradient, releasing free energy that fuels ATP synthesis in the mitochondrial matrix.

If the three ETC sites in the cristal membrane that actively transport protons are proton pumps, then the cristal membrane ATP synthase complexes function as regulated proton gates which catalyze ATP synthesis as protons flow through. For their discovery of the details of ATP synthase function, P. D. Boyer and J. E. Walker shared the Nobel Prize in Chemistry in 1997.

165-2 Proton Gates Capture Proton Gradient Free Energy as ATP

The ratio of ATP to ADP concentrations regulates proton flow through the ATP synthase gates. A high ATP/ADP ratio in the mitochondrial matrix indicates that the cell does not need more ATP and closes the proton gate so that the proton gradient cannot be relieved. On the other hand, a low ATP/ADP ratio in the matrix means that the cell is hydrolyzing a lot of ATP and that the cell needs more. Then the proton gate opens, and protons flow through cristal membrane ATP synthases back into the matrix, going down the concentration gradient. As they flow, they release free energy to power a protein motor in the enzyme, which in turn activates ATP synthesis. Just as we did for glycolysis, we can count the ATPs and see how much free energy we get from aerobic respiration, (i.e., the complete oxidation of glucose). You can see this in the following link.

166 A Balanced Sheet for Respiration

CHALLENGE

While warm-blooded animals keep nearly constant above ambient body temperatures, their cells may be hotter yet! Mitochondria work at temperatures up to \(50^{\circ} C\) (Hot Mitochondria.) How then might you account for our normal \(37^{\circ} C\) body temperatures?

CHALLENGE

Some experiments suggest that C. elegans longevity is assisted by normal mitochondrial ATP synthase activity. Other experiments suggest that the curcumin in turmeric, rejuvenates aging mouse and fly cells by targeting their ATP synthase! What does this spicy news suggest to you? (See ATP Synthase-Aging-Alzheimer’s Disease).

If the endosymbiotic theory is correct, then aerobic bacteria are the evolutionary ancestor to mitochondria, and the bacterial cell membrane should be the site an ETC and a chemiosmotic mechanism of ATP generation, much like that in mitochondria. This is in fact the case. And proton gradients not only power molecular motors linked to ATP synthesis but are also linked directly to the spinning of a bacterial flagellum (yet another “splendid molecular machine”, Figure 7.3, below.

Figure 7.3: A bacterial electron transport system can pump protons and other cations (Na+, Ca++) from the cytoplasm into the periplasmic space, at the left on the inner (cell) membrane. Relief of the proton gradient can power an ATP synthase or motility by fueling a spinning flagellum (at the right). In both cases. proton flow into the cell powers complex protein motors.

Electron transport in the cell membrane creates the gradient, in some cases cotransporting a proton and another cation (\(\rm Na^{+}\), \(\rm Ca^{++}\)). Relief of the proton gradient typically powers both ATP synthesis and the flagellum, though recent studies indicate that relief of the Na+ gradient can also spin the flagellum in some species of bacteria.