A high-potential periplasmic cytochrome from Sideroxydans lithotrophicus ES-1 with relevance to iron-oxidation pathways.

Abstract

Sideroxydans lithotrophicus (Sl) ES-1 is a neutrophilic Fe(II)-oxidizing bacterium (FeOB). The protein network involved in intracellular electron transfer in Sl and other neutrophilic FeOB is largely unknown. Our proteomics results reveal that a periplasmic monoheme cytochrome Slit_2042 is highly expressed and significantly more abundant when Sl cultures are grown on Fe(II) compared to thiosulfate. This pattern is similar to those for the iron oxidase Cyc2 and alternative complex III, suggesting the relevance of Slit_2042 in Fe(II) oxidation. We have developed a heterologous expression system to prepare Slit_2042 and characterized its structural and redox properties. Electronic absorption, 1H NMR, and EPR studies demonstrate that Slit_2042 has a six-coordinate, low-spin His/Met-ligated heme iron. The reduction potential of its heme iron is unusually high, 564 ± 2 mV. Analysis of the AlphaFold3 model of Slit_2042 reveals high structural similarity with two previously characterized cytochromes Hydrogenobacter thermophilus (Ht) c552 and Pseudomonas aeruginosa (Pa) c551. Molecular dynamics simulations of WT Slit_2042, W27F Slit_2042, Ht c552, and Pa c551 suggest that the high potential of the heme iron in Slit_2042 can be rationalized by the tight enclosure of the heme that arises from a unique β-hairpin surrounding heme propionates and a bulky Trp27 residue next to the His16 ligand. The high potential sets this cytochrome apart from other previously described cytochromes, and its relevance in the physiological function of Sl and design of protein-based systems for charge separation and catalysis is discussed.

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