Characterization of cytochrome 579, an unusual cytochrome isolated from an iron-oxidizing microbial community

A novel, soluble cytochrome with an unusual visible spectral signature at 579 nm (Cyt₅₇₉) has been characterized after isolation from several different microbial biofilms collected in an extremely acidic ecosystem. Previous proteogenomic studies of an Fe(II)-oxidizing community indicated that this abundant red cytochrome could be extracted from the biofilms with dilute sulfuric acid. Here, we found that the Fe(II)-dependent reduction of Cyt ₅₇₉ was thermodynamically favorable at a pH of >3, raising the possibility that Cyt₅₇₉ acts as an accessory protein for electron transfer. The results of transmission electron microscopy of immunogold-labeled biofilm indicated that Cyt₅₇₉ is localized near the bacterial cell surface, consistent with periplasmic localization. The results of further protein analysis of Cyt₅₇₉, using preparative chromatofocusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed three forms of the protein that correspond to different N-terminal truncations of the amino acid sequence. The results of intact-protein analysis corroborated the posttranslational modifications of these forms and identified a genomically uncharacterized Cyt₅₇₉ variant. Homology modeling was used to predict the overall cytochrome structure and heme binding site; the positions of nine amino acid substitutions found in three Cyt₅₇₉ variants all map to the surface of the protein and away from the heme group. Based on this detailed characterization of Cyt₅₇₉, we propose that Cyt₅₇₉ acts as an electron transfer protein, shuttling electrons derived from Fe(II) oxidation to support critical metabolic functions in the acidophilic microbial community.