Abstract
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 O2 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 FeII species, and an unassigned residual quadrupole doublet. The iron in stationary-phase cells was dominated by magnetically ordered FeIII 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) FeII pool consisting of two iron complexes with masses of ∼500 (major) and ∼1300 (minor) Da. They appeared to be high-spin FeII 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 O2 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 FeII pool from reacting with O2 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.
Highlights
Iron is critical for virtually all organisms, yet major questions remain regarding the systems-level understanding of iron in whole cells
The word content refers to the concentration of iron in such cells but to a semiquantitative description of the major iron species contained therein. 57Fe-enriched cells were grown on minimal medium using two different carbon sources, three different nutrient iron concentrations, and variable levels of O2 exposure
Our motivation was to gain a foundational understanding of iron trafficking and regulation in WT E. coli, as our previous MB investigations of whole cells have focused on eukaryotic systems
Summary
Iron is critical for virtually all organisms, yet major questions remain regarding the systems-level understanding of iron in whole cells. Using MB spectroscopy, Hristova et al [31] reported a fourth quadrupole doublet in whole E. coli cells, representing ϳ60% of spectral intensity in their samples This doublet was attributed to [Fe4S4]2ϩ and [Fe2S2]2ϩ clusters, low-spin FeII hemes, and possibly fast-relaxing high-spin FeIII species. Previous MB studies have decomposed the iron content of E. coli into four major groups These include magnetically ordered FeIII (which may represent the iron core of the ferritin FtnA), two nonheme high-spin FeII species (some of which may represent the LIP), and a group of overlapping iron centers that include [Fe4S4]2ϩ and [Fe2S2]2ϩ clusters, low-spin FeII hemes, and possibly fast-relaxing high-spin FeIII species. Our results suggest a major reinterpretation of iron homeostasis in this organism, and they reveal an unexpected and intriguing connection to the iron content of mitochondria and other prokaryotes
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