FERROUS IRON OXIDATION BY FERROBACILLUS FERROOXIDANS: PURIFICATION AND PROPERTIES OF Fe++-CYTOCHROMEcREDUCTASE

Abstract

The electron transport chain and Fe++-cytochrome c reductase of Ferrobacillus ferrooxidans were studied to elucidate the mechanism of iron oxidation by this autotrophic bacterium.The iron oxidation involved the cytochrome c and a type cytochrome of F. ferrooxidans in the electron transport system. The initial enzyme of the oxidation system was found to be Fe++-cytochrome c reductase. The iron oxidase system was labile to freezing or sonication; either treatment disrupted some link between the cellular cytochromes c and a. Fe++-cytochrome c reductase and cytochrome oxidase retained their individual activities after either treatment.Fe++-cytochrome c reductase was purified 60-fold to 70-fold. The enzyme was judged to be approximately 90% pure by disc electrophoresis, sedimentation, and DEAE-cellulose chromatography. A suitable assay system with a veronal–acetate buffer was developed for the determination of enzyme activity. The effects of inhibitors and potential activators were studied. No specific inhibitor or cofactor was found, although the enzyme was inhibited by various ionic compounds.Fe++-cytochrome c reductase was dissociated into two subunits, one protein and the other ribonucleic acid (RNA). Neither of the subunits had enzymatic activity and efforts to reconstitute the holoenzyme from the two subunits were unsuccessful. The molecular weights of the holoenzyme, protein subunit, and RNA subunit were determined as 100,000–110,000, 27,000–30,000, and 315,000–330,000, respectively. The protein subunit contained one non-heme iron atom per protein molecule. It was concluded that RNA is an essential component of the enzyme and the failure to recover the activity from subunits is due to the aggregation of RNA after dissociation.