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Reconstitution of Phosphorylating Electron Transport in Mitochondria from a Cytochrome c-deficient Yeast Mutant

From the ¹ From the Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and the Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14620

Electron transport and oxidative phosphorylation in mitochondria isolated from a cytochrome c-deficient mutant of the yeast, Saccharomyces cerevisiae, may be efficiently reconstituted by addition of purified yeast or horse heart cytochrome c. Added cytochrome c is tightly bound and is retained during reisolation of mitochondria.

Reconstituted mutant mitochondria carry out oxidative phosphorylation with substrates linked to nicotinamide adenine dinucleotide, flavoprotein, and cytochrome c as efficiently as mitochondria isolated from wild type yeast under the same conditions.

Titration of mutant mitochondria with yeast cytochrome c leads to progressive increases in P:O ratios as well as to increases in rates of phosphate and oxygen uptake. The end points of such titrations indicate that reconstitution is stoichiometric at a ratio of 0.2 mµmole of cytochrome c per mg of mitochondrial protein, approximately the amount of cytochrome c in normal wild type mitochondria.

Respiration in mutant mitochondria can be restored equally well by two types of yeast cytochrome c (iso-1- and iso-2-cytochrome c) and by horse heart cytochrome c. The two types of yeast cytochrome c are virtually equivalent in restoring phosphorylation coupled to succinate oxidation, whereas the horse heart enzyme is less effective.

Mutant mitochondria reconstituted with iso-1-cytochrome c catalyze phosphorylating oxidation of succinate, -ketoglutarate, glutathione plus tetramethyl-p-phenylenediamine, pyruvate plus malate, and d-lactate plus malate with efficiency comparable to that of normal yeast mitochondria. Ethanol oxidation was partially restored. Oxidative phosphorylation with l-lactate, even in the presence of added malate to provide citric acid cycle intermediates, was poorly restored by either of the isocytochromes c.

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