State IV Respiration

The energy in any mitochondrial substrate (succinate, glutamate, malate + pyruvate, fatty acids) is channeled to the ETS by a the reduced form of a 'high energy intermediate,' either NAD or FAD. Transport of electrons and free energy proceeds, and the electrons plus some free eneregy are used by complex IV, the cytochrome oxidase complex, to reduce oxygen. Experimentally, the decline in total dissolved oxygen as oxygen is reduced to water can be used to measure the rate of electron transport. Reduction of oxygen produces water, but the significance of oxygen reduction is that it removes electrons from the system. Electrons must be removed so that the carriers can be re-oxidized and continue to transport and store free energy.

If the chemiosmotic gradient builds up, why can electron transport proceed at all? A great many processes exploit the gradient, and some of them will be described below. However, in vitro the reason that we see a significant state IV respiration rate is the presence of damaged mitochondria in the preparation. Every isolated mitochondria preparation includes some mitochondria that cannot maintain a chemiosmotic gradient. If the outer membrane is stripped or the inner membrane is compromised, then in the presence of substrate electron transport runs freely, at the maximum possible rate. We refer to such mitochondria as uncoupled, because without a gradient electron transport cannot be coupled to ATP synthesis. So, in isolated mitochondria only, part of the state IV rate is due to damaged mitochondria that exercise no respiratory control at all.

In vivo the chemiosmotic gradient constantly decays due to a number of mechanisms. For example, the proton gradient is exploited by transport proteins in the inner membrane, in order to bring molecules into the matrix. Most important is the phosphate anion symport system that brings inorganic phosphate into the matrix for the phosphorylation of ADP. Exploitation of the gradient to bring in substances uses some of the energy, which is replaced by electron transport at a rate equal to the rate of loss of energy. Part of the rate of electron transport both in living cells and in isolated mitochondria can be attributed to symport systems.

By the way, there is no such thing as state IV respiration in vivo. Mitochondria in living tissues always conduct oxidative phosphorylation at rates that are proportional to the availability of ADP. ATP synthesis drains energy from the gradient by a specific route (see state III respiration). Part of the definition of state IV respiration is the absence of ADP.

Steady states

When we add the first substrate to isolated mitochondria we see a change in the rate of oxygen consumption followed by a steady state. Recall that in biological terms a steady state involves expenditure of energy to maintain some condition in a constant state. State IV respiration is a steady state because energy from the substrate is exploited to maintain a chemiosmotic gradient. The condition of the mitochondria remains constant as they deplete the medium of oxygen.

What is it that determines the rate of oxygen consumption in the steady state that we call state IV respiration? Earlier it was explained that the availability of substrate in these kinds of experiments is not limiting. Could some step in the sequence of events from substrate to the final oxygen reduction reaction be responsible for determining the rate? Whatever the answer, we know that in a sequence of events only one step can be rate-limiting.

When we add ADP to mitochondria that are in state IV respiration, oxygen consumption (electron transport) speeds up. The reason will be discussed in the article on state III respiration. If the ETS can speed up in the presence of ADP, could any part of the electron transport process have been responsible for limiting the rate in state IV? The answer is no. In respiratory control it is the rate of removal of energy from the gradient that determines how fast the ETS can transport electrons. The ETS will move electrons as fast as it is permitted to move them, up to the point at which some step in the process actually does prevent further acceleration of transport. Undamaged, unpoisoned mitochondria never reach that point.

When you analyze a process to determine what controls its rate, it is sometimes easier to rule out possibilities than to arrive at the answer directly. In state IV respiration the ETS is restricted by the gradient. It can speed up. Thus, no step leading up to or within the electron transport sequence can be rate-limiting. It must be the rate of energy loss from the gradient that determines the rate of oxygen consumption in state IV.

Cause and effect in state IV

The concept of respiratory control might be difficult to understand at first. Electron transport speeds up spontaneously whenever any restriction on it is removed. Each carrier down the chain is more electronegative than the preceding carrier. If there is substrate and oxygen, then the chemical reactions and transfers of electrons will take place. Remove all restrictions, and the ETS transports electrons at some maximum rate determined by type of substrate(s) available, physical conditions (temperature, etc.), and the number of available enzymes and ETS complexes. When energy is removed from the gradient at a faster pace the ETS speeds up spontaneously. It does not "want" to speed up, nor is it driven by a "need" to maintain the gradient. How could a need drive a physical process? As hard as it is, you must provide physically meaningful explanations for phenomena such as respiratory control.

Fatty acids can be used as substrates for studying isolated mitochondria. Energy from metabolism of fatty acids is delivered to Krebs cycle as acetyl coenzyme A, and the first high energy carrier to be reduced along the pathway is NAD. When we recover mitochondria through differential centrifugation, the concentrated pellet contains dissolved components of the original homogenate including fatty acids. Liver is a rather fatty tissue, thus the fatty acid concentration may be sufficient with which to support some respiration. However, although we may see oxygen consumption when we add mitochondria alone to a dissolved oxygen (oxygraph) chamber, they usually do not maintain a chemiosmotic gradient. We can check by adding a small amount of ADP to see if ADP can accelerate respiration. If not, then the rate of delivery of energy from the fatty acids is insufficient with which to keep up with the rate at which energy would be lost from a chemiosmotic gradient.

Respiration on glutamate, for example, is slower than respiration on succinate. Why? For electrons to be donated to the ETS, glutamate and NAD must both bind a matrix enzyme, glutamate dehydrogenase. Glutamate is oxidized to alpha-ketoglutarate and NAD is reduced to NADH in the process. NADH must then diffuse to complex I to donate its electron pair. Could the diffusion step be responsible for the slower rate on glutamate? Maybe. Again, think about what must follow from such a conclusion. If the rate of diffusion of NADH to complex I was rate limiting, could adding ADP cause respiration to speed up? No.

Electron transport from NADH to oxygen stores more energy than does electron transport from succinate to oxygen. Trace the respective pathways to see for yourself. The rate of removal of energy from the gradient regulates the rate of electron transport. Thus in any state in which respiratory control is maintained, it is the efficiency with which energy is replaced that determines which substrate produces a faster rate of oxygen consumption. So, which is more efficient – respiration on glutamate or respiration on succinate?

What happens if we destroy the gradient (see the article on poisons)? Again, think it through. With no gradient there can be no respiratory control. Now the ETS is unrestricted. Considering the diffusion step along the NADH pathway, which substrate is likely to produce the greatest rate of respiration, and why?