Evidence that a respiratory shield in Escherichia coli protects a low-molecular-mass FeII pool from O2-dependent oxidation

Abstract

Edited by Ruma Banerjee Iron is critical for virtually all organisms, yet major questions remain regarding the systems-level understanding of iron in whole cells. Here, we obtained Mössbauer and EPR spectra of Escherichia coli cells prepared under different nutrient iron concentrations, carbon sources, growth phases, and O 2 concentrations to better understand their global iron content. We investigated WT cells and those lacking Fur, FtnA, Bfr, and Dps proteins. The coarse-grain iron content of exponentially growing cells consisted of iron-sulfur clusters, variable amounts of nonheme high-spin Fe II species, and an unassigned residual quadrupole doublet. The iron in stationary-phase cells was dominated by magnetically ordered Fe III ions due to oxyhydroxide nanoparticles. Analysis of cytosolic extracts by size-exclusion chromatography detected by an online inductively coupled plasma mass spectrometer revealed a low-molecular-mass (LMM) Fe II pool consisting of two iron complexes with masses of ϳ500 (major) and ϳ1300 (minor) Da. They appeared to be high-spin Fe II species with mostly oxygen donor ligands, perhaps a few nitrogen donors, and probably no sulfur donors. Surprisingly, the iron content of E. coli and its reactivity with O 2 were remarkably similar to those of mitochondria. In both cases, a "respiratory shield" composed of membrane-bound iron-rich respiratory complexes may protect the LMM Fe II pool from reacting with O 2. When exponentially growing cells transition to stationary phase, the shield deactivates as metabolic activity declines. Given the universality of oxidative phosphorylation in aerobic biology, the iron content and respiratory shield in other aerobic prokaryotes might be similar to those of E. coli and mitochondria.

Ferric uptake regulator (Fur) reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis in Escherichia coli

“…The bacterial intracellular "free" iron concentration is primarily regulated by a global transcription factor Ferric uptake regulator (Fur) (1)(2)(3)(4). It has been generally assumed that when the intracellular "free" iron concentration is elevated, Fur binds "free" ferrous iron and the iron-bound Fur represses the genes encoding for iron uptake systems and stimulates the genes encoding for iron storage proteins (5)(6)(7)(8)(9).…”

A New Route for Dioxygen Activation Uncovered from Quantum Mechanics Investigations of X‐Ray‐Diffraction‐Captured Intermediates of the Ferroxidase Reaction of Ferritins from Gram‐Negative Bacteria

“…It is there that a dioxygen molecule can be entrapped, bringing about the oxidation of Fe II to Fe III hydrated oxo species which can migrate toward the central cavity. This way, excess cellular Fe II is prevented, while a reserve is also provided of Fe III (albeit in a much lesser relative amount than previously thought [ 5 ] ), which can be reduced back to Fe II when needed. [ 6,7 ]…”

Energy metabolism, oxygen flux, and iron in bacteria: The Mössbauer report

“…For example, quadrupole splitting occurs when the angular momentum quantum number, I, is Ͼ1/2 and is in a nonspherical electric field typical of an asymmetric ligand environment. Wofford et al (4) use this splitting as an important diagnostic in their survey of cellular iron species. These elements of Mössbauer spectra are illustrated by some of their most significant data in Fig.…”

“…There are several unique take-home messages provided by the data presented by Wofford et al (4). Previous work had provided evidence both for and against the storage of large amounts of iron as Fe III in ferritin (i.e.…”

“…A significant fraction of this iron was converted to what the authors have characterized as magnetically ordered ferric oxyhydroxide nanoparticles, importantly differing from previous assignments of this species as ferritin iron. Based on biochemical fractionation, the authors further linked this Fe II LMM species to what is commonly referred to as the cellular labile iron pool (4). The demonstration that this iron pool is sensitive to autoxidation and deposited as inert iron nanoparticles is a novel finding.…”