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Electron Transport Systems of the Chemoautotroph Ferrobacillus ferrooxidans

II. PURIFICATION AND PROPERTIES OF A HEAT-LABILE IRON-CYTOCHROME c REDUCTASE

From the ¹ From the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218

A heat-labile iron-cytochrome c reductase was isolated from Ferrobacillus ferrooxidans by sonic oscillation of the cells at pH 5.5 in the presence of protamine sulfate and purified 20-fold with 10% recovery by successive fractionation of the high speed (144,000 x g) supernatant solution with ammonium sulfate and Sephadex G-200.

The enzyme displayed a pH optimum of 5.7 to 6.2 and typical substrate saturation curves with a K_m of 2.75 x 10^-5 m for cytochrome c and 1.6 x 10^-3 m for FeSO₄·7H₂O. The protein nature of the catalyst was established by its protein content and susceptibility to the proteolytic action of Pronase and to heat. Exposure for 1 min at 100° was sufficient to destroy the activity completely. The most highly purified fraction of iron-cytochrome c reductase contained cytochrome c and apparently nonfunctional cytochrome b. Enzymatic activity, proportional to protein concentration and linear with time, was inhibited by reduced glutathione, p-hydroxymercuribenzoate, and cysteine, but not by atebrin, Amytal, or antimycin A. The inhibition by p-hydroxymercuribenzoate could be entirely reversed by equivalent concentrations of reduced glutathione or cysteine. Reduced cytochrome c, ferric ions, and several other metal ions also inhibited the reductase activity.

The close association of the enzyme with the iron oxidase activity of F. ferrooxidans was partially shown by the generally parallel distribution of the two activities in the various fractions resulting from Sephadex treatment and from differential centrifugation. Various attempts to separate the iron oxidase and cytochrome c reductase activities from one another in the less purified fractions were unsuccessful leading to destruction of either both activities or the iron oxidase activity. The most highly purified iron-cytochrome c reductase fraction (F₅) possessed no iron oxidase activity and contained a factor that stimulated less purified fractions several-fold in both their cytochrome c oxidase and iron oxidase activities but not in their iron-cytochrome c reductase activity.

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