Mitochondria In Vitro: Krebs cycle

When mitochondria are isolated for study they are usually depleted of substrates, with the exception of a low concentration of fatty acids from the tissues that were homogenized. The mitochondria are analagous to empty power plants, waiting for fuel. When we toss them into a respiration chamber they immediately take up oxygen into the matrix, noticeably reducing the oxygen concentration in the chamber. The initial steep decline in oxygen concentration is followed by a much slower rate that represents actual oxygen consumption, using fatty acids as a source of electrons and free energy.

Now suppose you add glutamate (glutamic acid). The glutamate dehydrogenase reaction takes place in the mitochondria matrix, oxidizing glutamate to alpha-ketoglutarate and reducing NAD to NADH. The NADH dehydrogenase complex on the inner membrane (complex I of the ETS) re-oxidizes NADH and electron transport continues from there.

If we are to use intact mitochondria to study respiratory control we need them to establish and maintain a chemiosmotic gradient unless they are deliberately poisoned. To accomplish that purpose we add a an excess of substrate so that during the course of oxygen consumption the mitochondria never run out. Availability of substrate is not rate-limiting.

After glutamate is converted to alpha-ketoglutarate, the latter compound is oxidized to succinyl-Coenzyme A, right? Technically, that statement is correct. Every product of a Krebs reaction can be further modified by another Krebs enzyme, so that the same carbon skeleton can "cycle" through all of the Krebs reactions. However during the course of an experiment so little alpha-ketoglutarate is produced that the rate of the second reaction can be considered to be negligible compared with the rate of the first reaction. The second reaction, therefore, is product dependent, that is, for the 'next' reaction to work, the product alpha-ketoglutarate must reach and bind alpha-ketoglutarate dehydrogenase. By adding glutamate alone to isolated mitochondria we can focus on the consequences of the glutamate dehydrogenase reaction (fate of NADH), and need not be concerned with the effects of additional Krebs reactions on oxygen consumption.

As an experiment proceeds, enough of the next product may be produced to begin to affect oxygen consumption. On glutamate, though, the next reaction also produces NADH. If electron transport is limited solely by the rate at which energy is removed from the chemiosmotic gradient, then the second reaction will not affect oxygen consumption qualitatively or quantitatively.

When researchers wish to isolate the succinate dehydrogenase reaction they usually include rotenone in the respiration medium. Rotenone blocks respiration on NADH but not on succinate. With rotenone present even if subsequent Krebs reactions produce a significant amount of NADH, oxygen consumption will not be affected. In the teaching lab our experiments go quickly enough that we don't measure any contribution by NADH when we use succinate as sole substrate, even without using rotenone. For example, our ADP:O ratios on succinate are usually 2/3 that of the ratios on glutamate, that is, close to 2 as opposed to close to 3.

Of course, even if the reactions could cycle all the way to oxaloacetate, a source of acetyl coenzyme A would be needed in order to regenerate citric acid. In fact, one combination of substrates that is used by investigators is malate + pyruvate. As an exercise, see if you can figure out how malate + pyruvate might affect oxygen consumption. Trace the path of energy to the electron transport system.

Key concept

• For any substrate added to isolated mitochondria, you only need to be concerned with the first energy carrier that comes from the Krebs reaction(s). Rates of subsequent reactions depend on the concentrations of products of the preceding reactions, and occur much more slowl.