A sulphate metabolizing centre in Euglena mitochondria

Abstract

We have previously shown that a sulphate activating system is present on the outside of the inner mitochondrial membrane of Euglena gracilis Klebs. var. bacillaris Cori, but efforts to couple this system to ATP produced from oxidative phosphorylation were unsuccessful. In the present work we show that the concentration of Pi ordinarily used to support oxidative phosphorylation in these mitochondria (10 mM) inhibits sulphate activation completely; by reducing the concentration of Pi 10-fold, both processes proceeded normally. Sulphate activation under these conditions is inhibited nearly completely by the uncouplers of oxidative phosphorylation dinitrophenol (0.1 mM) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) (0.2 microM). Sulphate reduction to form free cysteine, most of which appears outside the organelle, and in the cysteine of mitochondrial protein can be demonstrated in the same preparations, is membrane-bound and is inhibited by chloramphenicol (100 micrograms/ml), NaN3 (5 mM), KCN (100 microM); dinitrophenol (0.1 mM) or CCCP (0.2 microM). Digitonin fractionation of the mitochondria into mitoplasts, outer membranes and an intermembrane fraction show that reduction of 35SO4(2-) to form free cysteine and cysteine of protein is located on the mitoplasts; adenosine 5'-phosphosulphate sulphotransferase, the first enzyme of sulphate reduction, is found in the same location. Sulphate activation is highly enriched in the mitochondrial fraction of Euglena; the small amount found in the chloroplast fraction can be attributed to mitochondrial contamination. Thus, in Euglena, sulphate activation and reduction are contained in a sulphate metabolizing centre on the outside of the mitochondrial inner membrane; this centre appears to supply the mitochondrion and the rest of the cell with the products of sulphate activation as well as with reduced sulphur in the form of cysteine. Mitochondria from wild-type Euglena cells and from W10BSmL, a mutant lacking plastids completely, appear to be similar in the properties studied.