Superoxide-mediated amplification of the oxygen-induced switch from [4Fe-4S] to [2Fe-2S] clusters in the transcriptional regulator FNR

Abstract

In Escherichia coli, the switch between aerobic and anaerobic metabolism is controlled primarily by FNR (regulator of fumarate and nitrate reduction), the protein that regulates the transcription of >100 genes in response to oxygen. Under oxygen-limiting conditions, FNR binds a [4Fe-4S]2+ cluster, generating a transcriptionally active dimeric form. Upon exposure to oxygen the cluster converts to a [2Fe-2S]2+ form, leading to dissociation of the protein into monomers, which are incapable of binding DNA with high affinity. The mechanism of cluster conversion together with the nature of the products of conversion is of considerable current interest. Here, we demonstrate that [4Fe-4S]2+ to [2Fe-2S]2+ cluster conversion, in both native and reconstituted [4Fe-4S] FNR, proceeds via a one electron oxidation of the cluster, to give a [3Fe-4S]1+ cluster intermediate, with the release of one Fe2+ ion and a superoxide ion. The cluster intermediate subsequently rearranges spontaneously to form the [2Fe-2S]2+ cluster, with the release of a Fe3+ ion and, as previously shown, two sulfide ions. Superoxide ion undergoes dismutation to hydrogen peroxide and oxygen. This mechanism, a one electron activation of the cluster, coupled to catalytic recycling of the resulting superoxide ion back to oxygen, provides a means of amplifying the sensitivity of [4Fe-4S] FNR to its signal molecule.

Fig. 1.

Predicted structure of a FNR monomer. The proposed structure of a FNR monomer, based on homology with CRP. The locations of important features are shown. The model was generated by using Swiss-Model and the Swiss-PDB viewer (9) with CRP as a template (7).

Fig. 2.

Detection of an intermediate cluster during the oxidation of [4Fe-4S] FNR. (A) EPR spectra of reconstituted [4Fe-4S] FNR (20.4 μM), in buffer A, as a function of time after exposure to oxygen (219.5 μM, 21°C). EPR parameters: temperature, 15 K; microwave power, 2.0 mW; frequency, 9.67 GHz; modulation amplitude, 0.5 mT. Spectra are normalized to the same gain. (B) Correlation of EPR observations with optical (420 nm) observations. Optical data are indicated in gray, and EPR data are indicated as filled circles. Double-exponential function fits of the optical and EPR data are shown as a solid line and broken line, respectively (see Data Analysis). Fitting parameters, k_obs1 = 0.0611 s⁻¹, k_obs2 = 0.0087 s⁻¹.

Fig. 3.

Detection of superoxide during [4Fe-4S] FNR oxidation. Cytochrome c reduction measured at 21°C in the presence of reconstituted [4Fe-4S] FNR (≈9 μM), oxidized cytochrome c (74 μM) and oxygen (219.5 μM), oxygen and catalase (CAT), or oxygen and SOD (dashed black line), as indicated. Anaerobic (dashed gray line) and anaerobic with CAT and SOD (solid gray) served as controls. Reactions were carried out in buffer A. Single exponential fits are drawn in (solid black lines).

Fig. 4.

Oxygen dependence of cluster conversion measured by optical absorbance. (A) Reconstituted [4Fe-4S] FNR (5 μM) was mixed with aliquots of buffer B containing varying concentrations of dissolved oxygen at 25°C. Loss of the [4Fe-4S] cluster was monitored at 420 nm as a function of time (in gray). Double- exponential function fits of the data are indicated by solid black lines. Arrows indicate the direction of response with increasing initial oxygen concentrations. (B) Plot of the first observed (first order) rate constants obtained from the data in A and similar experiments as a function of oxygen concentration (filled circles). A least-squares linear fit (black line) of the data is drawn in.

Fig. 5.

Oxygen dependence of the rate of Fe²⁺ release from [4Fe-4S] FNR upon oxidation. (A) Reconstituted [4Fe-4S] FNR (2 μM) was mixed with aliquots of buffer B containing varying concentrations of dissolved oxygen at 25°C. Release of Fe²⁺ was monitored with Ferene (100 μM) at 593 nm (in gray). Single- exponential function fits of the data are indicated by solid black lines. Arrows indicate the direction of response with increasing initial oxygen concentrations. (B) Plot of the first observed (first order) rate constants obtained from the data in A and similar experiments as a function of oxygen concentration (filled circles, left ordinate). A least-squares linear fit (black line) of the data is drawn in. The dependence of Fe²⁺ recovered per [4Fe-4S] by Ferene is plotted as a function of the oxygen concentration (open triangles, right ordinate).