Iron proteins and enzymes serve essential and diverse functions—from central metabolism to DNA regulation—in most living cells. ⁵⁷Fe-Mössbauer spectroscopy facilitates quantitative and qualitative probing of the structure of iron proteins, often being the only technique to provide insights into difficult electronic structure assignments. Whole-cell Mössbauer spectroscopy can sometimes be used to bridge the gap between results obtained on an isolated purified protein and the hard-to-achieve picture of the working protein in the cellular milieu. In addition to its occasional utility in obtaining in vivo mechanistic evidence, Mössbauer has also been used to study the ironome, providing an iron-centric view of the cell.
The goal of this presentation is to critically examine whole-cell Mössbauer spectroscopy, exploring its strengths and limitations through the lens of fundamentals of the technique. We aim to present basic parameters such resolution, signal-to-noise, linewidths, and evaluate how whole-cell spectra would stand being tested by the same standards as their isolated-protein counterparts. As a case study we use our own whole-cell spectra of the ferric uptake regulator, Fur. In an effort to better understand the structure of iron cofactors in the Fur protein, we employed whole-cell Mössbauer spectroscopy. Previously we showed that iron-bound Fur isolated from E. coli bacteria contains one [Fe₂-S₂]²⁺ cluster per monomer, bound in a site different form the Zn binding site. Whole-cell Mössbauer spectra of E. coli containing the H. influenzae and E. coli Fur protein and control cells were collected in multiple experimental conditions. The difference spectra were analyzed and were unambiguously identified with the Fur protein containing an [Fe₂-S₂]²⁺ cluster.
In a comparative analysis, we aim to explore what standards of resolution and spectral fitting and what expectations would be appropriate in the interpretation for whole-cell spectra, given the global nature of iron pools and signal overlaps that are inevitable in the examination of cells.

Thomas Hoepner

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