title: Characterization of cytochrome 579, an unusual cytochrome isolated from an iron-oxidizing microbial community.
abstract: A novel, soluble cytochrome with an unusual visible spectral signature at 579 nm (Cyt(579)) 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 Cyt(579) acts as an accessory protein for electron transfer. The results of transmission electron microscopy of immunogold-labeled biofilm indicated that Cyt(579) is localized near the bacterial cell surface, consistent with periplasmic localization. The results of further protein analysis of Cyt(579), 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 Cyt(579) 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 Cyt(579) variants all map to the surface of the protein and away from the heme group. Based on this detailed characterization of Cyt(579), we propose that Cyt(579) acts as an electron transfer protein, shuttling electrons derived from Fe(II) oxidation to support critical metabolic functions in the acidophilic microbial community.
authors.affiliation: Chemistry Directorate, Lawrence Livermore National Laboratory, Livermore, CA 94550, USA.
authors.forename: Steven W
authors.initials: SW
authors.lastname: Singer
authors.forename: Clara S
authors.initials: CS
authors.lastname: Chan
authors.forename: Adam
authors.initials: A
authors.lastname: Zemla
authors.forename: Nathan C
authors.initials: NC
authors.lastname: VerBerkmoes
authors.forename: Mona
authors.initials: M
authors.lastname: Hwang
authors.forename: Robert L
authors.initials: RL
authors.lastname: Hettich
authors.forename: Jillian F
authors.initials: JF
authors.lastname: Banfield
authors.forename: Michael P
authors.initials: MP
authors.lastname: Thelen
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D018441:Biofilms; D003580:Cytochromes / Q000737:chemistry* / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005583:Fourier Analysis; D007501:Iron / Q000378:metabolism*; D013058:Mass Spectrometry; D046529:Microscopy, Electron, Transmission; D008906:Mining; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid
chemical_list: D003580:Cytochromes; D007501:Iron