The Bradyrhizobium japonicum frcB Gene Encodes a Diheme Ferric Reductase

Abstract

Iron utilization by bacteria in aerobic environments involves uptake as a ferric chelate from the environment, followed by reduction to the ferrous form. Ferric iron reduction is poorly understood in most bacterial species. Here, we identified Bradyrhizobium japonicum frcB (bll3557) as a gene adjacent to, and coregulated with, the pyoR gene (blr3555) encoding the outer membrane receptor for transport of a ferric pyoverdine. FrcB is a membrane-bound, diheme protein, characteristic of eukaryotic ferric reductases. Heme was essential for FrcB stability, as were conserved histidine residues in the protein that likely coordinate the heme moieties. Expression of the frcB gene in Escherichia coli conferred ferric reductase activity on those cells. Furthermore, reduced heme in purified FrcB was oxidized by ferric iron in vitro. B. japonicum cells showed inducible ferric reductase activity in iron-limited cells that was diminished in an frcB mutant. Steady-state levels of frcB mRNA were strongly induced under iron-limiting conditions, but transcript levels were low and unresponsive to iron in an irr mutant lacking the global iron response transcriptional regulator Irr. Thus, Irr positively controls the frcB gene. FrcB belongs to a family of previously uncharacterized proteins found in many proteobacteria and some cyanobacteria. This suggests that membrane-bound, heme-containing ferric reductase proteins are not confined to eukaryotes but may be common in bacteria.

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

“…Irr is present and active under iron limitation, and genes under positive control are involved in iron acquisition and other functions that allow adaptation to this environment. Irr represses genes encoding proteins that function optimally under iron-replete conditions, including those that contain iron, synthesize heme and iron-sulfur clusters, and respond to high-iron stress (210,274,318,(324)(325)(326)(327)(328)(329)(330)(331)(332).…”

Vibrio cholerae VciB Mediates Iron Reduction

“…Both B. japonicum (30) and Listeria monocytogenes (43,44) use reductases to increase the amount of ferrous iron that can be transported for use by the bacterial cell. The Bradyrhizobium reductase FrcB resembles proteins of the cytochrome b superfamily and mediates iron reduction through two heme prosthetic groups (30). Small and O'Brian (30) were able to purify FrcB to demonstrate heme association and iron-dependent oxidation of the heme groups in vitro.…”

“…His38 localizes within the membrane, and His166 is in the periplasmic loop of the protein. Histidine residues may be required for binding cations (49), binding a cofactor (30), or aiding in electron transfer to ferric iron in concert with tyrosine (50). Most of the known iron reductases use heme to reduce the ferric iron (30,37,51).…”

“…The reduction assay was modified from Small et al (30) as follows. Strains bearing the indicated plasmids grown on LB agar plates were inoculated into LB medium with appropriate antibiotics and grown overnight.…”

“…Many of the characterized bacterial reductases involved in iron acquisition are soluble and use cofactors (32). The Bradyrhizobium japonicum FrcB reductase is a membrane-associated cytochrome b-type protein that requires heme for reductase activity (30). For ferric iron reductases that allow iron to serve as the terminal electron acceptor, such as those studied in Shewanella oneidensis (33)(34)(35), Geobacter sulfurreducens (36,37), and E. coli (34), electrons from the oxidation of NADH are passed through the electron transport chain to iron.…”