Direct nitric oxide signal transduction via nitrosylation of iron-sulfur centers in the SoxR transcription activator

Abstract

Nitric oxide (NO) has diverse roles in intercellular communication and (at higher levels) in immune-mediated cell killing. NO reacts with many cellular targets, with cell-killing effects correlated to inactivation of key enzymes through nitrosylation of their iron-sulfur centers. SoxR protein, a redox-sensitive transcription activator dependent on the oxidation state of its binuclear iron-sulfur ([2Fe-2S]) centers, is also activated in Escherichia coli on exposure to macrophage-generated NO. We show here that SoxR activation by NO occurs through direct modification of the [2Fe-2S] centers to form protein-bound dinitrosyl-iron-dithiol adducts, which we have observed both in intact bacterial cells and in purified SoxR after NO treatment. Functional activation through nitrosylation of iron-sulfur centers contrasts with the inactivation typically caused by this modification. Purified, nitrosylated SoxR has transcriptional activity similar to that of oxidized SoxR and is relatively stable. In contrast, nitrosylated SoxR is short-lived in intact cells, indicative of mechanisms that actively dispose of nitrosylated iron-sulfur centers.

Figure 1

Rapid activation of SoxR in NO-exposed E. coli. (A) Transient induction of soxS by brief NO exposure. Growing cultures of strain XA90 with the SoxR-expression plasmid pKOXR were treated to induce SoxR and then were transferred to anoxic conditions. Five-milliliter aliquots of anoxic culture were mixed with 100 μl of NO-saturated water (≈2 mM), with the mixture completed within 10 sec. Anoxic incubation was continued, and samples were withdrawn at the times indicated in the figure to analyze for soxS mRNA by Northern blotting. (B) Extended soxS induction by extended NO exposure. Samples were generated as for A, except that the NO solution was added over ≈45 sec. (C) Quantitation of soxS induction by NO. Northern blots as in A and B were quantitated by phosphorimaging.

Figure 2

EPR spectroscopy of nitrosylated SoxR in intact cells. (A) E. coli XA90/pKOXR (21) were grown to OD₆₀₀ ≈1 and were concentrated 25-fold. The concentrated cells (1 ml) were mixed with 0.2 ml of a NO-saturated solution in water (as described in Fig. 1A) and after 2 min were frozen at −170°C. EPR spectroscopy of the intact cells was carried out as described (21). The g values used to model the EPR signals are indicated (g = 1.92, reduced [2Fe-2S] SoxR; g = 2.03, NO-SoxR). (B) Kinetics of nitrosyl-SoxR formation and removal in intact cells. XA90/pKOXR cells were exposed to NO as indicated in Fig. 1A. At the indicated times after NO addition, 0.4-ml aliquots were collected into EPR tubes and immediately were frozen at −170°C. EPR analysis of these cells was as described (21). The profiles shown are the net signal after subtraction of spectra for control cells not overexpressing SoxR. (C) Correlation of nitrosyl-SoxR and transcription in intact cells. The amplitude of the g = 2.03 signal was measured and is plotted in comparison with the soxS mRNA level in the same cells (Fig. 1C).

Figure 3

NO modification of SoxR in vitro. Purified SoxR protein (23, 33) (2.5 ml, 3 μM) was mixed with increasing amounts of a NO-saturated solution in water to yield the indicated final concentrations of NO (nominal; some is lost to the gas phase and some by reaction with residual oxygen or other components present in the sample). The samples were frozen and subjected to EPR spectroscopy. The inset shows the quantitation of the amplitude of the g = 2.03 signal as a function of NO treatment.

Figure 4

Properties and activity of re-purified, NO-treated SoxR. (A) EPR spectroscopy. Purified SoxR protein (23, 33) (2.5 ml, 3 μM) was mixed with 100 μl of a NO-saturated solution in water. The NO-treated protein was then reduced with sodium dithionite (20 mM), followed by reoxidation with 1 mM potassium ferricyanide. Trace a, dithionite-reduced, purified SoxR; trace b, NO-treated SoxR, showing d7 signal; trace c, NO-treated SoxR reduced with dithionite, showing d9 signal; trace d, reoxidation of protein from trace c. A small signal at ≈3,500 G may be attributable to dithionite, as it persists in the reoxidized sample (compare traces c and d). (B) In vitro transcription by NO-treated SoxR. In vitro transcription reactions (23, 24) used the indicated amounts of purified, oxidized SoxR (SoxR [2Fe-2S]), or purified, NO-treated SoxR (SoxR Fe-NO). The transcripts were quantified by primer extension (40). bla, SoxR-independent transcript; soxS, transcript from the SoxR-dependent soxS gene.

Figure 5

Distinct activation mechanisms for SoxR. Shaded ovals: DNA binding domains; unshaded ovals, iron-binding domains. SoxR is shown as a homodimer (16). (A) Redox-regulation. When the [2Fe-2S] centers of SoxR are in the reduced state, the protein does not activate soxS transcription. One-electron oxidation converts the protein to a potent transcription activator. (B) Model for activation of SoxR by nitrosylation. The intact [2Fe-2S] clusters of SoxR are directly modified by NO to generate dinitrosyl-iron-dithiol clusters. This modification disrupts the iron-sulfur clusters; the remaining thiols are provided by the cysteine residues of SoxR protein. The model shows all iron atoms remaining bound to the protein, but may not be the case, particularly in the protein subjected to repurification.