Mössbauer spectroscopy as a tool for the study of activation/inactivation of the transcription regulator FNR in whole cells of Escherichia coli - PubMed (original) (raw)

Mössbauer spectroscopy as a tool for the study of activation/inactivation of the transcription regulator FNR in whole cells of Escherichia coli

C V Popescu et al. Proc Natl Acad Sci U S A. 1998.

Abstract

The global regulator FNR (for fumarate nitrate reduction) controls the transcription of >100 genes whose products facilitate adaptation of Escherichia coli to growth under O2-limiting conditions. Previous Mössbauer studies have shown that anaerobically purified FNR contains a [4Fe-4S]2+ cluster that, on exposure to oxygen, is converted into a [2Fe-2S]2+ cluster, a process that decreases DNA binding by FNR. Using 57Fe Mössbauer spectroscopy of E. coli cells containing overexpressed FNR, we show here that the same cluster conversion also occurs in vivo on exposure to O2. Furthermore, the data show that a significant amount of the [4Fe-4S]2+ cluster is regenerated when the cells are shifted back to an anaerobic environment. The present study also demonstrates that 57Fe Mössbauer spectroscopy can be employed to study the in vivo behavior of (overexpressed) proteins. The use of this technique to study other iron-containing cell components is discussed.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Mössbauer spectra at 4.2 K of purified 4Fe-FNR (A), of 2Fe-FNR (B), and of E. coli whole cells containing overexpressed FNR (FNR+ sample), exposed for 15 min to air (D, dash marks). Spectra of A and B were adapted from figure 1 of ref. . The solid line in D is a “background” spectrum (mostly because of ferritin) obtained by subtracting the doublet of A from the anaerobic FNR+ sample (sample of Fig. 2_B_, spectrum not shown), assuming that 4Fe-FNR represents 14% of total Fe. The difference spectrum (C, dash marks), which represents the FNR species in the air-exposed cells, was obtained by subtracting the “background” spectrum (D, solid line) from that of the air-exposed sample (D, dash marks). The solid line in C is a theoretical curve obtained by adding the doublets of 4Fe-FNR and 2Fe-FNR according to a 1:1 cluster ratio, i.e., 2:1 in Fe. The arrow indicates the Fe2+ released during cluster conversion; the vertical dotted line marks the high-energy line of the [2Fe-2S]2+ cluster. The spectra shown in D are from the same samples as those of Fig. 2_C_.

Figure 2

Figure 2

Mössbauer spectra of E. coli cells recorded at 1.5 K (A–C) and 4.2 K (E). (A) FNR− cells, anaerobic (dash marks) and after exposure to air (solid line). (B) Anaerobic FNR+ cells. Bracket marks 4Fe-FNR. (C) Anaerobic FNR+ cells from B (dash marks) and FNR+ cells exposed for 15 min to air (solid line). (D) Difference spectrum of the anaerobic FNR+ sample minus the spectrum of the air-exposed FNR+ sample, by using the spectra shown in C. The solid line in D is a theoretical difference spectrum assuming 4Fe-FNR and 2Fe-FNR in 1:1 cluster ratio. (E) Whole-cell spectra of FNR-L28H mutant, anaerobic (dash marks), and exposed to air for 20 min (solid line).

Figure 3

Figure 3

Mössbauer spectra at 4.2 K of FNR+ cells (batch different from that in Figs. 1 and 2). Spectra of anaerobic cells (A, dash marks), air-exposed cells (A and B, solid line) and cells after removal of air and incubation under argon at 4°C (B, dash marks). The arrow marks the high-energy absorption feature of Fe2+, released by cluster conversion.

Similar articles

Cited by

References

    1. Beinert H, Holm R H, Münck E. Science. 1997;277:653–659. - PubMed
    1. Johnson M K. Curr Opin Chem Biol. 1998;2:173–181. - PubMed
    1. Guest J R, Green J, Irvine A S, Spiro S. In: Regulation of Gene Expression in Escherichia coli. Lin E C C, Lynch A S, editors. Austin, TX: Landes; 1996. pp. 317–342.
    1. Lazazzera B A, Beinert H, Khoroshilova N, Kennedy M C, Kiley P J. J Biol Chem. 1996;271:2762–2768. - PubMed
    1. Khoroshilova N, Popescu C, Münck E, Beinert H, Kiley P. Proc Natl Acad Sci USA. 1997;94:6087–6092. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources