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

Abstract

Abstract 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, α-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.