Sub-mitochondrial particles


Treatment of mitochondria with ultrasonic vibrations under appropriate conditions tears open the membranes and leads to the formation of tiny inside-out vesicles derived from the inner membrane, containing trapped cytochrome c originating from the inter-membrane space. Sub-mitochondrial particles may retain partial coupling between electron transport and ADP phosphorylation. They have been much used for experimental work because their inverted membrane orientation provides direct access to the respiratory chain without the complications introduced by the substrate transport systems.

Two activities which are most easily studied in sub-mitochondrial particles (although they also occur with intact mitochondria) are the energy linked transhydrogenase and reversed electron transport.

The energy-linked transhydrogenase: This bizarre membrane-spanning enzyme catalyses the reversible transfer of reducing equivalents between the mitochondrial NADPH + NADP pool and the NADH + NAD pool, while simultaneously pumping 2 protons and 2 charges across the inner mitochondrial membrane. The energy linkage keeps the NADPH/NADP couple about 500 times more reduced than the NADH/NAD couple, despite the exact equivalence of their standard redox potentials.

NADPH + NAD+ + 2H+(inside) <=> NADP+ + NADH + 2H+(outside)

It is not clear which direction the transhydrogenase takes in vivo. Enzymes reacting with NADP generally have a more negative redox potential than those reacting with NAD, except for glutamate dehydrogenase, which is the only enzyme with a dual coenzyme specificity. This is a problem, since the dual coenzyme specificity should lead to an energy-wasting futile cycle if the two coenzyme pools are at different redox potentials.

The arrangement must confer some selective advantage (having persisted unchanged for 2000 million years!) but the biological benefits remain obscure. It may involve the overall regulation of nitrogen balance, by using the ammonia-fixing reaction with NADPH to partially counteract the ammonia formation with NAD, but this hypothesis remains highly speculative. Mitchell once suggested that the NADPH-linked pathways for isocitrate, malate and glutamate were equivalent to fifth gear on a car, giving enhanced ATP yields and better fuel economy at the expense of snappy performance. When maximum ATP flux was required the ADP-mediated activation of the NAD-linked pathways would "drop down a gear" to enter the overtaking lane.

Reversed electron transport: Oxidative phosphorylation is a partially reversible process and in the presence of an artificially high ATP/ADP ratio electrons from a weak reducing agent like succinate can be forced backwards through the respiratory chain carriers to yield a stronger reductant such as NADH:

succinate + NAD+ + energy => fumarate + NADH + H+     (overall reaction)

Ethanol is relatively poor reducing agent, and will only reduce a tiny proportion of any added NAD. However, in an artificial system containing sub-mitochondrial particles, added alcohol dehydrogenase, a trace of NAD and excess NADP, electrons from ethanol can be forced backwards via a small pool of NADH through the energy-linked transhydrogenase to form large amounts of the excellent reducing agent, NADPH. The reaction requires a source of energy: either added ATP, or respiration using another segment of the respiratory chain.

ethanol + NAD+ => acetaldehyde + NADH + H+

NADH + NADP+ + energy => NAD+ + NADPH

The process has little physiological relevance, but gave enormous insight into mitochondrial function. Reversed electron transport from ethanol to NADP in rotenone-blocked sub-mitochondrial particles can be driven either by energy from external ATP (reversing the normal operation of the F1ATPase) or by the energy from succinate oxidation via complex 2, complex 3 and complex 4. Both routes are sensitive to uncouplers, but oligomycin only blocks the process when it is driven by ATP. This key observation showed that there must be a common high energy intermediate between the coupling sites on the respiratory chain and the manufacture of ATP. The intermediate is known as the high energy pool.

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