RECONSTITUTION OF MITOCHONDRIAL RESPIRATORY CHAIN

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The aim of this research is to obtain basic information on the respiratory chain enzyme system of mitochondrial inner membrane enabling conclusions on carriers, their sequence, actions and mechanisms of electron-hydrogen transport (which eventually leads to energy coupling). The methods planned will be mainly by sequential resolution, systematic reconstitution, enzyme identification, and related techniques. Among others, the resolved enzymes and components are first purified (preferably crystallized, if possible) and then rigorously characterized by various physicochemical and enzymic techniques, such as rapid and "slow" methods for kinetics, amino acid sequence analysis, magnetic resonance techniques, EXAFS, etc. It should be emphasized that reconstitution will be used as a most reliable technique (at present) to ascertain that no denaturation or changes occurred in the extensive purification of components in order to obtain clear-cut results. An ultimate goal in our first phase, not necessarily the eventual one, is reconstitution with well defined respiratory enzymes or components of the complete electron transport chain which constitutes a part of bioenergetic apparatus with all functional manifestations of the mitochondrion. It must be pointed out that this application (which differs from another project) emphasizes and deals with aspects on cytochrome-containing parts of the respiratory chain, except components most intimately associated with cytochromes.