The Dark Side of Respiratory Chain: Superoxide Formation | Aging and Cell Death

An Electron Transport Chain is a series of coupled reactions taking place between electron donors and electron acceptors. This causes an electrochemical gradient to be produced in the membrane which is later used to derive chemical energy in the form of Adenosine Triphosphate (ATP).

Most of the oxygen consumed by aerobic organisms happens to get reduced to water by the enzyme cytochrome c oxidase in the terminal reaction of the ETC take takes place in the inner mitochondrial membrane. Since when oxygen is in the ground state, it acquires a triplet configuration (with two unpaired electrons in the outer shell), its reduction to water must have to occur in the four major consecutive one-electron steps. Some of the partially reduced oxygen intermediates generated in this process happen to be very stable but cytochrome c oxidase is able to retain them until the process gets over and all the electrons are transferred. Although, a small proportion of the oxygen molecules [1-2% (Boveris et al., 1973)] happen to get converted to superoxide anion radical typically by other respiratory components. There are basically two major sites in the respiratory chain where these reactions happen to take place: Complex I [NADH dehydrogenase, (Turrens et al., 1980)] and Complex III [ubisemiquinone, (Boveris et al., 1976; Cadenas et al., 1977; Turrens et al., 1985)].

Before dwelling into the typical mechanism of superoxide formation, the basic cycle of the electron transport chain should be kept into mind. The electron transport is carried out by four complexes namely NADH dehydrogenase also known as NADH:ubiquinone oxidoreductase, succinate dehydrogenase, cytochrome bc1 complex and cytochrome c oxidase. The NADH for the initiation of the process is derived from the products of Citric Acid Cycle also known as Kreb's cycle. NADH transfers two of its electrons to ubiquinone which gets reduced to ubiquinone (QH2) that gets diffused into the membrane causing complex 1 to translocate 4 protons (H+) across the membrane thus creative a proton flux (complex I). Further, additional electrons are transferred to Q via FAD from succinate in complex II. In Complex III, asymmetric distribution of electrons takes place. Two electrons are taken from the Q0 site of Quinone (Q) and then transferred to cytochrome C. In complex IV, four electrons are removed from four molecules of cytochrome c and transferred to molecular oxygen (O2) thus leading to the formation of water (H2O). The electrochemical gradient produced during this series of events is used to generate energy in the form of ATP.

Although when electron transfer is reduced, Complex I and III may leak electrons to molecular oxygen, resulting in superoxide formation. This reduction in electron transfer may be caused by a high membrane potential or respiratory inhibitors such as antimycin A. Complex I is one of the major sites where premature electron leakage to oxygen occurs which could be fatal.

Complex I is a potent source of reactive oxygen species. It can produce superoxide as well as hydrogen peroxide through at least two different pathway. During forward ETC, the production of superoxide is very low, nearly 0.1% of the total electron flow occurring. While in the reverse flow of electron in which electron transport occurs in a reverse direction to reduce NAD to NADH, the rate of superoxide formation is very high as 5% of the electron flow is directed towards complex I. Studies by Muller FL et al. shows that in vitro during reverse flow of electrons, when succinate levels are high and oxaloacetate or malate concentration is low, complex I can be a very potent source for superoxide formation. Superoxide is a highly reactive species that leads to cellular oxidative stress and neuromuscular disease & aging. The ratio of NADH to NAD+ determines the rate of superoxide formation.

Complex III is another site in ETC where premature electron leakage occurs resulting in the formation of superoxide which is linked to several pathologies and aging as per the free radical aging theory. The electron leakage occurs mainly at the Q0 site and is stimulated by antimycin A. The mechanism behind it is that antimycin A inhibits re-oxidation of b hemes at Qi site leaving them in reduced state. This causes the concentrations of the Qo semiquinone to rise and its reaction with oxygen to ultimately form superoxide. The superoxide produced at Qo site can either be released into intermembrane space from where it can reach into cytosol or mitochondrial matrix or both.

Effect of inhibitors on superoxide formation during ETC
Since the superoxide formation by complex I and III in the electron transport chain or respiratory chain is a non-enzymatic process (equation 1), this activity happens to increase by a mass action either when the electron carriers are highly reduced or when there is a presence of mitochondrial inhibitors. Superoxide production by mitochondrial electron carriers also increases with oxygen concentration as can be inferred from equation 1. This is due to cytochrome c oxidase which when has a Km for oxygen in the sub-micromolar range, is typically saturated under physiological conditions and is not expected to compete for the extra oxygen.

d[Superoxide]/dt = k[O2] x [XH] -Equation (1)

One example of the effect of mitochondrial inhibitors is Antimycin A which has been mentioned before. Others includes rotenone, myxothiazol, cynide etc. Addition of inhibitors into the picture, however, makes it much more complex.

Thus, while on one hand the ETC contribute to cell-growth and cell survival by producing chemical energy in the form of ATP derived from the electrochemical gradient across the inner mitochondrial membrane, the premature leakage of oxygen at the major two sites viz. complex I and II leading to formation of harmful superoxide could be fatal and contributes to many pathological factors including aging and cell death. A small proportion of the oxygen utilized by aerobic cells happen to undergo partial reduction by Electron transport chain components, however the toxic effects of these reactive species is prevented by antioxidant defences. Although, under pathologic conditions, these defences fail leading to oxidative stress and then eventually damaging mitochondria and other cell compartments, ultimately leading to cell death.

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