Oxidation of cysteine to cystine by membrane fractions of Chlorella fusca.

Isolated membrane fractions of Chlorella fusca 211-8b obtained by french-press treatment and sonication catalyzed the oxidation of L-cysteine to L-cystine. The pH-optimum of this reaction was determined to be around 8-8.5 and a stoichiometry of 4 SH-groups oxidized for one O2 consumed was obtained. This thiol-oxidation system was specific for D-and L-cysteine; DL-homocysteine and cysteamine were oxidized at about half the rate whereas all other thiols tested including glutathione, mercaptoethanol, mercaptopropionic acid and dithioerythritol were not oxidized by these membrane fractions. The apparent Km for L-cysteine was determined as 3.3 mmol l(-1). Rates of 200 μmol cysteine oxidized mg(-1) chlorophyll h(-1) were normally obtained. Extremely high rates of oxygen uptake were measured using L-cysteine methyl ester and L-cysteine ethyl ester. This thioloxidation system was not inhibited by mitochondrial electron-transport inhibitors such as rotenone or antimycin A, nor by the chloroplast electron-transport inhibitors 2,5-dibromothymochinone and 2,4-dinitrophenylether of iodonitrothymol. The cysteine oxidation catalyzed by C. fusca membranes was inhibited, however, by salicylhydroxamic acid, o-phenanthrolin, N,N'-disalicyliden-1,3-diaminopropane 5,5'-disulfonic acid, ethylenediaminetetraacetic acid, high KCN levels and by the buffers, N-[2-hydroxyl-1,1-bis(hydroxymethyl) ethyl] glycine and phosphate. This cysteine-oxidation system seems to function as a counterpart of thioredoxin-mediated light activation of enzymes, allowing reduced thiol groups to be oxidized again by O2 (dark inactivation).