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Applied and Environmental Microbiology, July 2008, p. 4454-4462, Vol. 74, No. 14
0099-2240/08/$08.00+0     doi:10.1128/AEM.02799-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of Cytochrome 579, an Unusual Cytochrome Isolated from an Iron-Oxidizing Microbial Community{triangledown}

Steven W. Singer,1 Clara S. Chan,2,{dagger} Adam Zemla,3 Nathan C. VerBerkmoes,4 Mona Hwang,1 Robert L. Hettich,4 Jillian F. Banfield,2 and Michael P. Thelen1*

Chemistry Directorate,1 Computations Directorate, Lawrence Livermore National Laboratory, Livermore, California 94550,3 Department of Earth and Planetary Sciences, University of California, Berkeley, Berkeley, California 94720,2 Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 378314

Received 11 December 2007/ Accepted 26 March 2008

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 Cyt579 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.

* Corresponding author. Mailing address: Lawrence Livermore National Laboratory, L-452, P.O. Box 808, Livermore, CA 94551-0808. Phone: (925) 422-6547. Fax: (925) 422-2282. E-mail: mthelen{at}llnl.gov

{triangledown} Published ahead of print on 9 May 2008.

{dagger} Present address: Woods Hole Oceanographic Institution, Woods Hole, MA 02543.

Applied and Environmental Microbiology, July 2008, p. 4454-4462, Vol. 74, No. 14
0099-2240/08/$08.00+0     doi:10.1128/AEM.02799-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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