Iron oxidation by cell envelopes of Thiobacillus ferrooxidans

Abstract

The parameters for iron oxidation by isolated cell envelopes of Thiobacillus ferrooxidans were studied using a Clark oxygen electrode. Envelopes were obtained by breaking thoroughly washed cells in a French pressure cell and by sedimenting the envelope fraction by ultracentrifugation. The envelopes were partially purified by sucrose density-gradient centrifugation. A broad band was separated at a specific gravity of about 1.21 g/cm3 which coincided with the major protein peak and iron oxidation activity. Electron micrographs confirmed that the active preparations were cell envelopes consisting of the cytoplasmic membrane, peptidoglycan layer, and the outer lipopolysaccharide–lipoprotein double track layer of gram-negative bacteria. Numerous membrane vesicles were also present. Lysozyme had little effect upon iron oxidation activity whereas treating cell envelopes with Triton X-100 destroyed activity. Iron oxidation by cell envelopes was completely inhibited by heating in boiling water or by adding 1% trichloroacetic acid to the reaction cuvette.Kinetic analysis of iron oxidation by envelopes in β-alanine-SO42− buffer (pH 3.5) showed an apparent Km of 5.4 × 10−3 M. The apparent Km with whole cells was 2.1 × 10−3 M. The pH optimum for iron oxidation by cell envelopes was between pH 3.0 and 3.5 while for whole cells the optimum was pH 2.0–2.5.Spectrophotometric studies identified the membrane-bound cytochromes as cytochromes c and a; cytochrome b was also present but its function is unknown at this time.