Cytochrome 572 is a conspicuous membrane protein with iron oxidation activity purified directly from a natural acidophilic microbial community

Abstract

Recently, there has been intense interest in the role of electron transfer by microbial communities in biogeochemical systems. We examined the process of iron oxidation by microbial biofilms in one of the most extreme environments on earth, where the inhabited water is pH 0.5-1.2 and laden with toxic metals. To approach the mechanism of Fe(II) oxidation as a means of cellular energy acquisition, we isolated proteins from natural samples and found a conspicuous and novel cytochrome, Cyt 572, which is unlike any known cytochrome. Both the character of its covalently bound prosthetic heme group and protein sequence are unusual. Extraction of proteins directly from environmental biofilm samples followed by membrane fractionation, detergent solubilization and gel filtration chromatography resulted in the purification of an abundant yellow-red protein. The purified protein has a cytochrome c-type heme binding motif, CxxCH, but a unique spectral signature at 572 nm, and thus is called Cyt 572. It readily oxidizes Fe 2 in the physiologically relevant acidic regime, from pH 0.95-3.4. Other physical characteristics are indicative of a membrane-bound multimeric protein. Circular dichroism spectroscopy indicates that the protein is largely beta-stranded, and 2D Blue-Native polyacrylamide gel electrophoresis and chemical crosslinking independently point to a multi-subunit structure for Cyt 572. By analyzing environmental genomic information from biofilms in several distinctly different mine locations, we found multiple genetic variants of Cyt 572. MS proteomics of extracts from these biofilms substantiated the prevalence of these variants in the ecosystem. Due to its abundance, cellular location and Fe 2 oxidation activity at very low pH, we propose that Cyt 572 provides a critical function for fitness within the ecological niche of these acidophilic microbial communities. © 2008 International Society for Microbial Ecology All rights reserved.