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& Data Analysis


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Mitochondria theory

Mitochondria in vitro

Additional topics


State III Respiration

State III respiration is defined as ADP-stimulated respiration. We put isolated mitochondria into state III when we add ADP to intact, unpoisoned mitochondria in the presence of excess substrate. That is, to produce state III we must first produce state IV. The mechanism for acceleration of respiration in the presence of ADP was illustrated in the introduction. ADP binds the enzyme complex ATP synthase. In the presence of inorganic phosphate, which also binds ATP synthase, ADP binding opens a channel that permits the driving of protons into the matrix from outside the inner membrane. The energy that is released as protons are driven across is used to produce ATP. As energy in the gradient is removed the electron transport chain spontaneously speeds up. The synthesis of ATP by mitochondria is called oxidative phosphorylation.

It must be emphasized that ATP synthase is not part of the ETS, although it does float around in the same lipid bilayer as components of the ETS. It must also be emphasized that the protons that pass through ATP synthase simply recombine with hydroxyl ions in the matrix. They are ionization products of water in this system as they are in any aqueous system. There is nothing special about these particular protons. They do not drive reactions, and they certainly do not reduce oxygen. Oxygen consumption in state III is caused by the same process as is oxygen consumption in state IV. It is catalyzed by cytochrome oxidase as the last step in electron transport.

In an isolated system, the mitochondria return to state IV respiration when the ADP is used up. The state IV rate may be higher than before, due to an uncoupling effect by ADP itself.

Uncoupling effect of ADP

Following state III respiration the state IV rate that resumes after ADP is used up is often greater than the initial state IV rate. The observation can be bothersome when one's research calls for reporting the R.C.R. (Respiratory Control Ratio, the ratio of state III to state IV respiration). Having a probable explanation can be reassuring.

In living cells the ratio of ATP to ADP is quite high. The concentration of ADP in a cell never approaches that found in a dissolved oxygen chamber when an investigator squirts a pile of it in with a syringe. I'll offer two possible suggestions for the mechanism by which ADP may cause an increase in electron transport.

Perhaps there is a nonspecific effect of ADP on the membrane itself (perhaps the outer, perhaps the inner, maybe both). That is, in large concentrations ADP may bind proteins or lipids in the membrane, creating a channel of sorts or otherwise disrupting the structure so that protons can move across. I sort of prefer a more elegant explanation, however. All living membranes must be maintained constantly by energy-requiring reactions. In fact, one reason death occurs so quickly following cyanide poisoning is that the supply of ATP is completely cut off. Cells need a constant, immediate supply of ATP. Without it their membranes begin to deteriorate immediately. Nerves, in particular, stop conducting right away, and the victim is history. If the ratio of ATP to ADP is so unfavorable that 'housekeeping' reactions are stopped or significantly slowed, there may be damage to the mitochondrial membranes. Most likely, some mitochondria become totally uncoupled while others are able to maintain a gradient.

ADP:Oxygen Ratio

The passage of an electron pair through each of the proton-translocating complexes (I, III, and IV) is associated with a drop in free energy that somewhat exceeds that needed to phosphorylate one molecule of ADP. The phosphorylation of one mole of ADP requires 30.5 kJ (kilojoules). The free energy released on the passage of two moles of electrons (they move in pairs, you know) is 51 kJ through complex I, 41 kJ through complex III, and 100 kJ through complex IV. Since it is only at these three sites that electron transport releases enough free energy to phosphorylate ADP, it was hypothesized that the passage of one pair of electrons from NADH through the length of the ETS causes the phosphorylation of three molecules of ADP (two molecules if the starting point is FADH2).

In the early 1970s, three major hypotheses were kicking around, to explain the coupling of electron transport to ADP phosphorylation. The term coupling refers to how the energy released through electron transport is transferred to ADP and inorganic phosphate. One proposal called for a chemical intermediate to carry energy to an ATP synthetase enzyme, much like NADH carries energy to the ETS from Krebs reactions. A second proposal called for a conformational change in a membrane-bound complex that is directly associated with each complex. The change in conformation would store energy, and when ADP and phosphate bound the complex the energy would be transferred. The last, the Mitchell hypothesis, was considered by many to be too far-fetched to be true. The proposal was that the energy was stored in the form of an electrochemical gradient, which was then utilized at a remote site to synthesize ATP.

Support for the first two proposals came in part from the observation that isolated mitochondria appear to produce three ATPs per electron pair via the "long" route, and 2 via the "short" (succinate) route. However, no one could demonstrate the presence of the putative chemical intermediate, and no one could match up binding sites for ADP and phosphate with any of the complexes of the ETS. It was observed that the extramitochondrial pH dropped when mitochondria were in the active state. Under ideal circumstances, isolated mitochondria could phosporylate slightly more than three ADPs per oxygen atom. These and other observations led to universal acceptance of Peter Mitchell's proposal and his eventual Nobel Prize.

You can verify that NADH-supported respiration results in the phosphorylation of 1.5 times as much ADP as with succinate-supported respiration by examining the ratio of ADP molecules phosphorylated to atoms of oxygen consumed (ADP:O ratio). You will likely observe that the ratios are not integers and that they are lower with uncoupled mitochondria and higher with well-coupled mitochondria. In fact, the ratio can exceed three in very well-coupled mitochondria (3.3 is about the maximum ratio attainable). That is because at the 'weakest' point of the chain, 41 kJ of free energy is available, and it only takes 30.5 kJ to phosphorylate one ADP. The theoretical maximum ADP:O ratio is not 4 because of the entropy associated with all processes, and because of utilization of the chemiosmotic gradient for other processes.

Calculating an ADP:O Ratio from a Chart Record

One can calculate an ADP:O ratio with minimal knowledge of the preparation itself. All you need is the total dissolved oxygen in the chamber from which you measured oxygen consumption, the total amount of ADP added to the chamber, and the percent of total oxygen that was used up in order to phosphorylate all of the ADP. Total oxygen is obtained by multiplying chamber volume by the known volume of dissolved oxygen per unit volume at the temperature of the experiment (0.237 micromoles molecular oxygen per ml at room temperature for a typical respiration medium).

Determine the amount of ADP added in µmoles or nmoles. Determine total oxygen in the chamber by multiplying chamber volume by the solubility factor in µmoles (or nmoles). Using a straightedge, define the slopes with light pencil lines and determine where the slopes intersect. Measure the percent of record (percent total oxygen) used during state III respiration by determining the distance between intersections at the start and end of state III. Convert to a fraction and multiply the fraction by total oxygen to get the amount of molecular oxygen used. Multiply by 2 to get the amount of atomic oxygen used. Divide the amount of ADP added by the amount of atomic oxygen consumed.


Let the chamber volume be 2 ml and solubility factor 0.237 micromoles/ml. Suppose the addition of 20 µl 0.01M ADP resulted in a 10% drop in total oxygen during state III respiration. Then 200 nmoles (0.2 µmoles) ADP was added to a chamber containing a total, at the beginning of the experiment, of 2 x 237 = 474 nmoles (0.474 µmoles) molecular oxygen. Molecular oxygen consumed was 0.10 x 474 = 47.4 nmoles (0.0474µmoles), which is 94.8 nmoles (0.0948 µmoles) atomic oxygen. The ADP:O ratio is given by 200 divided by 94.8, which is 2.1. Note that the ratio is reported to two significant figures, which is reasonable given the uncertainty of measuring volumes and the points at which the slope changed.

Report a realistic and appropriate ADP:O ratio

Students often report ADP:O ratios that are unrealistically precise. A ratio of 2.9885462, for example, suggests an impossible level of precision. Consider how accurately you can measure the slope of an oxygraph record before reporting ratios to several decimal places. Some students report ADP:O ratios for every experiment in which ADP was added to the chamber, even when state III respiration was not produced. If there is no state III, then there is no ATP synthesis and there cannot be an ADP:O ratio.

Students often report unrealistic values for ADP:O ratios. The quality of a preparation affects the ratio, but not so much that the ratio can be off by an order of magnitude. Even our best preparations will not produce ratios much above 3. Typical ratios for succinate and glutamate supported respiration approach 2 and 3, respectively.

Factors other than the condition of mitochondria affect ADP:O ratios. The amount of ADP added to a chamber determines how much oxygen will be consumed during the state III rate. In addition to the accuracy with which the ADP is drawn up and delivered, the concentration of the solution itself may be low or high. ADP deteriorates with time, so that a stored solution will be at lower concentration that when it was originally made up. On the other hand, a common student error is to thaw a stock solution and use it immediately without re-mixing the materials. As an aqueous solution freezes, the most dilute part freezes first. Since ice floats, the most concentrated part is at the bottom of the sample tube, and students usually go to the bottom when drawing liquid from a tube. It is critical to mix previously frozen stock solutions and samples before using them, usually by inverting and agitating the completely thawed solution repeatedly.

You may discover that upon the addition of an inhibitor of electron transport such as cyanide, that the chamber actually gains oxygen. Of course, there is no biochemical mechanism by which mitochondria evolve oxygen. The gain is from oxygen diffusing back into the chamber through a leaky seal or through the sample port. If such diffusion can occur at the end of the experiment, it certainly could take place (and was taking place) during the experiment itself.

Notice that some of the factors affecting ADP:O ratios tend to increase the measured ratios while others tend to decrease them. A discussion in a research paper should focus on the results themselves. Suppose one obtains an ADP:O ratio that is unusually low. A superficial discussion lists all of the factors that can affect ADP:O ratios without regard to which factors lower the measured ratio and which raise it. The reader will be interested in likely reasons for the observed result, not in every possible explanation for every possible observation.

Is entropy necessarily a bad thing?

In living cells mitochondria are never idle. They exercise respiratory control, with electron transport slowing or increasing as the energy in the chemiosmotic gradient is decreasingly or increasingly utilized. Much of the energy released during Krebs reactions and electron transport is converted to random molecular motion (heat). In fact, any reaction results in an increase in entropy of the universe (second law of thermodynamics). Should we despair this waste of valuable free energy?

We homeotherms maintain a constant internal temperature within physiological limits. Regulating body temperature requires regulating both heat production and heat loss. Mitochondria metabolism is an important source of heat production. For the most part, variations in the rate of electron transport are directly related to the demand by the cells for ATP. To some extent the rate also depends on the need to maintain a basal metabolic rate (BMR). We even have mechanisms for varying the extent to which mitochondria are uncoupled, so as to increase or decrease heat production on a long term basis.

Copyright and Intended Use
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Created by David R. Caprette (caprette@rice.edu), Rice University 12 Dec 96
Updated 27 May 05