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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.

SUBMITTER: Singer SW

PROVIDER: S-EPMC2493166 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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

Singer Steven W SW Chan Clara S CS Zemla Adam A VerBerkmoes Nathan C NC Hwang Mona M Hettich Robert L RL Banfield Jillian F JF Thelen Michael P MP

Applied and environmental microbiology 20080509 14

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, r ...[more]

PMID: 18469132

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Project description:Iron (Fe) oxidation is one of Earth's major biogeochemical processes, key to weathering, soil formation, water quality, and corrosion. However, our understanding of microbial contribution is limited by incomplete knowledge of microbial iron oxidation mechanisms, particularly in neutrophilic iron oxidizers. The genomes of many diverse iron oxidizers encode a homolog to an outer membrane cytochrome (Cyc2) shown to oxidize iron in two acidophiles. Phylogenetic analyses show Cyc2 sequences from neutrophiles cluster together, suggesting a common function, though this function has not been verified in these organisms. Therefore, we investigated the iron oxidase function of heterologously expressed Cyc2 from a neutrophilic iron oxidizer Mariprofundus ferrooxydans PV-1. Cyc2PV-1 is capable of oxidizing iron, and its redox potential is 208 ± 20 mV, consistent with the ability to accept electrons from Fe2+ at neutral pH. These results support the hypothesis that Cyc2 functions as an iron oxidase in neutrophilic iron-oxidizing organisms. The results of sequence analysis and modeling reveal that the entire Cyc2 family shares a unique fused cytochrome-porin structure, with a defining consensus motif in the cytochrome region. On the basis of results from structural analyses, we predict that the monoheme cytochrome Cyc2 specifically oxidizes dissolved Fe2+, in contrast to multiheme iron oxidases, which may oxidize solid Fe(II). With our results, there is now functional validation for diverse representatives of Cyc2 sequences. We present a comprehensive Cyc2 phylogenetic tree and offer a roadmap for identifying cyc2/Cyc2 homologs and interpreting their function. The occurrence of cyc2 in many genomes beyond known iron oxidizers presents the possibility that microbial iron oxidation may be a widespread metabolism. IMPORTANCE Iron is practically ubiquitous across Earth's environments, central to both life and geochemical processes, which depend heavily on the redox state of iron. Although iron oxidation, or "rusting," can occur abiotically at near-neutral pH, we find neutrophilic iron-oxidizing bacteria (FeOB) are widespread, including in aquifers, sediments, hydrothermal vents, pipes, and water treatment systems. FeOB produce highly reactive Fe(III) oxyhydroxides that bind a variety of nutrients and toxins; thus, these microbes are likely a controlling force in iron and other biogeochemical cycles. There has been mounting evidence that Cyc2 functions as an iron oxidase in neutrophiles, but definitive proof of its function has long eluded us. This work provides conclusive biochemical evidence of iron oxidation by Cyc2 from neutrophiles. Cyc2 is common to a wide variety of iron oxidizers, including acidophilic and phototrophic iron oxidizers, suggesting that this fused cytochrome-porin structure is especially well adapted for iron oxidation.

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Dataset Information

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

Publications

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

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