Brief history & orientation


Cytochrome spectra from the respiratory pigments in animal tissues were originally observed in the nineteenth century, but dismissed as an artefact by the scientific establishment of the day. Oxygen uptake by tissue homogenates, and the action of simple respiratory inhibitors were first studied systematically in the 1930s, when it was realised that electrons flowed from substrates to oxygen via a sequence of redox carriers, several of which showed distinctive spectral changes when oxidised and reduced.

The isolation of intact mitochondria followed improvements in centrifuge technology in the 1940s, and the introduction of isotonic sucrose preparation media to prevent osmotic lysis. Investigators noted the phenomenon of latency: the internal mitochondrial enzymes could not be demonstrated until the organelles had been broken open. The basic structure of mitochondria was established by electron microscopy and it was shown by controlled disruption that most of the respiratory enzymes responsible for oxygen uptake were attached to the inner mitochondrial membrane (which is extensively folded to form the mitochondrial cristae) while the soluble enzymes catalysing the Krebs cycle and fatty acid oxidation pathways were confined to the internal matrix space.

This electron microscope picture of a chick embryo mitochondrion is used by kind permission of Professor Ruth Bellairs, Department of Anatomy and Developmental Biology, University College, Gower Street, London WC1E 6BT.

It was later shown that the inner membrane was a major permeability barrier within the cell, but the outer membrane contained a protein called porin which rendered it largely largely permeable to molecules less than about 1500 daltons. An important group of enzymes which metabolise ATP, including myokinase, creatine kinase, and the nucleoside diphosphate kinases, are trapped in the inter-membrane space, between the inner and the outer membranes.

myokinase: ATP + AMP <=> ADP + ADP

creatine kinase: ATP + creatine <=> ADP + creatine phosphate

cytidine diphosphate kinase: CDP + ATP <=> CTP + ADP (many similar enzymes exist)

Experimental work accelerated with the introduction of the more convenient Clarke-type oxygen electrode in the 1950's in place of the older manometric methods. It was shown that the uptake of oxygen by intact mitochondria depended on the simultaneous conversion of ADP and inorganic phosphate into ATP. The phenomenon is termed respiratory control. The obligatory dependence of a highly favourable chemical reaction (substrate oxidation) on the simultaneous execution of an unrelated and extremely unfavourable reaction (ATP synthesis) was immediately recognised as a novel and important concept by the biochemists of the day.

Oxygen uptake which is dependent on the presence of ADP and phosphate is termed coupled respiration. The coupling of respiration and phosphorylation could be broken by mechanical or osmotic disruption of the mitochondria, or by the addition of uncoupling agents, in which case respiration proceeded rapidly without any concomitant ATP synthesis. The original uncoupling agent was 2,4-dinitrophenol, which has now been replaced by more effective compounds such as FCCP (para-trifluoromethoxy carbonyl cyanide phenylhydrazone) and CCCP (meta-chloro carbonyl cyanide phenylhydrazone).

Two major classes of mitochondrial inhibitor could be distinguished: respiration inhibitors and phosphorylation inhibitors. Both types were effective against intact mitochondria, but only respiration inhibitors could prevent oxygen uptake after the addition of an uncoupling agent. These experiments gave rise to the concept of an energetically favourable electron transport system, which in some way powered an energetically unfavourable phosphorylation system. The free energy available from the redox reactions was used to drive ATP synthesis. Respiration inhibitors such as cyanide ions blocked the electron transport chain and were always effective in suppressing oxygen uptake, whereas phosphorylation inhibitors such as oligomycin, which prevented ATP manufacture, could only block respiration if the coupling were intact.

From this point, in the mid 1950s, the subject diverted into a long and occasionally acrimonious blind alley, as scientists attempted to find a chemical intermediate linking respiration to ATP generation. This idea derived from the substrate level phosphorylations observed in the glycolytic pathway. This search was unsuccessful as no such intermediate exists. We now know that the energy from respiration is first captured in the form of pH and electrical potential gradients across the inner mitochondrial membrane. The energy stored in these gradients is later exploited to drive the synthesis of ATP. This chemiosmotic theory was first proposed in 1961 by a British biochemist, Peter Mitchell, who worked for many years without much official recognition or significant public funding until he was awarded the Nobel Prize for Chemistry in 1978.

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