Oxidative Phosphorylation of Mitochondrion (With Diagram)

Linkage of phosphorylation to oxidative metabolism was first proposed in the 1930s.

The first evidence was the observation that phosphate was removed from the medium when TCA cycle intermediates were me­tabolized by cells.

Subsequently, it was shown that the disappearance of inorganic phosphate was accompa­nied by the accumulation of organic phosphates such as glucose-6-phosphate and fructose-6-phosphate.

Shortly thereafter, ATP was recognized as the pri­mary product of oxidative phosphorylation. In 1948, Kennedy and Lehninger established that these oxida­tions and phosphorylation occurred exclusively in the mitochondria. By 1951, Lehninger had shown that the Krebs cycle reactions could be bypassed entirely by adding NADH and H⁺ to mitochondria treated to al­low the NADH to enter the organelles. For each NADH and H ⁺ added, three phosphates and one oxy­gen were consumed, and it was therefore concluded that the electron transport chain was important for ATP synthesis.

The potential differences of the reactions of elec­tron transfer beginning with NAD ⁺ -dehydrogenases and ending with cytochrome oxidase provide the en­ergy to drive the endergonic oxidative phosphoryl­ation. The exergonic reactions can be summarized as

NADH + H⁺ + ½O₂ + NAD⁺ + H₂O

Which has a ∆G⁰‘ of -220.5 kJ/mole (-52.7 kcal/ mole). The endergonic reactions summarized as

3 ADP + 3 P_i →3 ATP

are associated with a standard free energy of at least 91.6 kJ (i.e., ∆G⁰‘= 30.5 kJ/mole or 7.3 kcal/mole). The efficiency of the coupling of the two systems is at least 42% (i.e., 91.6/220.5). The stages at which coupling occurs are known. Figure 16-25 shows the major energy changes along the electron transfer chain. The step from NADH to Q represents the reactions of the dehydrogenases that link NAD ⁺ and F_P to Q.

This reaction sequence (Site I) provides enough energy (51.0 kJ/mole or 12.2 kcal/ mole) for the formation of the first of three ATPs ob­tained by NADH oxidation. The step from Q to cyto­chrome b does not provide enough energy for the second phosphorylation, but the following step (from cytochrome b to cytochrome c) does yield sufficient energy (Site II).

The step from cytochrome c to cyto­chrome a is only moderately exergonic, but the final step from cytochrome a to oxygen is highly exergonic (Site III). Thus, a pair of electrons transferred from one carrier to the next by these oxidation-reduction reactions at three places in the chain generates the energy for the formation of ATP.

However, not all elec­trons from oxidized substrates enter the electron transport chain at the initial NAD⁺ position. Some electrons, like those from succinate, pass from the co­enzyme of succinate dehydrogenase (i.e., FAD) di­rectly to Q. When this happens, only two ATPs are formed per pair of electrons transferred.

Molecular Events in Oxidative Phosphorylation:

The mechanism by which energy from the electron transport system is used for oxidative phosphoryl­ation is far from clear. It would appear that there are yet to be discovered coupling factors and enzymes in­volved in the process.

Among the more significant ob­servations that have been made are the identification of:

(1) Mechanisms for controlling the rate of electron transport and oxidative phosphorylation,

(2) Inhibi­tors that can prevent ATP formation or uncouple elec­tron transport from oxidative phosphorylation,

(3) Partial oxidative phosphorylation reactions, and

(4) Coupling factors.

Maximal electron transport in mito­chondria can occur only if there is ample ADP and P; available to act as an acceptor of inorganic phosphate.

When ADP is lacking or when ATP is plentiful, the rate of respiration is low and is called state 4 respira­tion (Fig. 16-26). When ADP is added, the rate of oxygen consumption rises as the ADP is phosphoryla- ted. This elevated level of respiration is called state 3 respiration. Once the ADP is consumed, state 4 respi­ration is reestablished.

The influence of ADP in respi­ratory rate is called acceptor or respiratory control, the “acceptor” being ADP. Interestingly, the configu­ration of mitochondria changes between state 4 and state 3 respiration. During state 4 respiration, the mitochondrion assumes the orthodox conformation but changes to the condensed conforma­tion when added ADP induces state 3.

A number of chemical agents uncouple oxidative phosphorylation from the electron transport system (e.g., 2, 4-dinitrophenol, dicumarol, and the salicyl- anilides). The addition of these compounds has two in­teresting effects. First, they speed up electron trans­port and oxygen consumption even in the absence of ADP, but there is no synthesis of ATP. In other words, phosphorylation is uncoupled and so is energy trans­fer. Second, in the presence of the uncoupling agent, the hydrolysis of ATP may occur—the opposite of the normal goals of mitochondrial activity.

Oligomycin is an inhibitor of oxidative phosphoryl­ation and coupled oxygen consumption but it does not prevent electron transfer in uncoupled systems. A group of agents called ionophores also prevent oxida­tive phosphorylation by dissipating the energy needed for phosphorylation. Ionophores are only effective in the presence of certain monovalent cations such as K⁺ and Na⁺.

Some of the reactions of oxidative phosphorylation are known. An ATPase is present in the inner mem­brane and is stimulated by 2, 4-dinitrophenol but inhib­ited by oligomycin. Another reaction is the exchange of inorganic phosphate with the terminal phosphate of ATP, a reaction called phosphate-ATP exchange. This reaction is inhibited by both 2, 4-dinitrophenol and oli­gomycin.

An exchange of atoms also occurs between the oxygen’s of water and those of inorganic phos­phate, called phosphate-water exchange. The terminal phosphate can also be exchanged between ATP and ADP, a process inhibited by 2, 4-dinitrophenol and oli­gomycin and called the ADP-ATP exchange reaction. Some interesting studies that relate structure and function in mitochondria have been carried out using inverted submit ochondrial vesicles formed from frag­ments of the inner membrane. In these studies, the in­ner membranes of mitochondria isolated from cells are subjected to sonification or detergent action.

The resulting fragments of the cristael membranes then round up to form vesicles with the inner membrane spheres on the outside surface (Fig. 16-27). If closed vesicles are formed, they exhibit functioning electron transport system and oxidative phosphorylation reac­tions. There is no ATP formation with open vesicles or non-vesicular fragments, but electron transport still occurs.

If the submitochondrial vesicles are treated with urea or trypsin, the inner membrane spheres are re­moved and can be separated from the vesicles, which lose their capacity to carry out oxidative phosphoryl­ation. However, if the spheres are added back to the vesicles, the capacity to perform oxidative phospho­rylation is regained. The spheres contain ATPase/ synthetase, which is called coupling factor one (F,). This enzyme has been purified and found to have a molecular weight of about 360,000, a diameter of 9 nm, and a requirement for Mg².

In intact mitochon­dria the enzyme is bound to the membrane and also catalyzes the synthesis of ATP and ADP and inor­ganic phosphate, rather than the reverse, and as such could be termed ATP-synthetase. The isolated en­zyme’s function, however, is not inhibited by oligomy­cin. Another factor has been isolated that when present with isolated F₁ renders the ATPase sensitive to oligomycin.

This latter factor is called F₀ or oligomycin-sensitivity-conferring protein (OSCP). F₀ is also a large protein; it is speculated that F₀ could constitute at least part of the stalk that attaches the sphere to the inner membrane.