The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion

Abstract

Fumarate and nitrate reduction regulatory (FNR) proteins are bacterial transcription factors that coordinate the switch between aerobic and anaerobic metabolism. In the absence of O(2), FNR binds a [4Fe-4S](2+) cluster (ligated by Cys-20, 23, 29, 122) promoting the formation of a transcriptionally active dimer. In the presence of O(2), FNR is converted into a monomeric, non-DNA-binding form containing a [2Fe-2S](2+) cluster. The reaction of the [4Fe-4S](2+) cluster with O(2) has been shown to proceed via a 2-step process, an O(2)-dependent 1-electron oxidation to yield a [3Fe-4S](+) intermediate with release of 1 Fe(2+) ion, followed by spontaneous rearrangement to the [2Fe-2S](2+) form with release of 1 Fe(3+) and 2 S(2-) ions. Here, we show that replacement of Ser-24 by Arg, His, Phe, Trp, or Tyr enhances aerobic activity of FNR in vivo. The FNR-S24F protein incorporates a [4Fe-4S](2+) cluster with spectroscopic properties similar to those of FNR. However, the substitution enhances the stability of the [4Fe-4S](2+) cluster in the presence of O(2). Kinetic analysis shows that both steps 1 and 2 are slower for FNR-S24F than for FNR. A molecular model suggests that step 1 of the FNR-S24F iron-sulfur cluster reaction with O(2) is inhibited by shielding of the iron ligand Cys-23, suggesting that Cys-23 or the cluster iron bound to it is a primary site of O(2) interaction. These data lead to a simple model of the FNR switch with physiological implications for the ability of FNR proteins to operate over different ranges of in vivo O(2) concentrations.

Fig. 1.

Activity of FNR proteins with Phe substitutions at residues adjacent to essential Cys ligands. Plasmid pGS24 was subjected to QuikChange site-directed mutagenesis (Stratagene) to create Phe substitutions and introduced to JRG1728 (fnr lac) containing pFF-41.5, a plasmid with an FNR-activated promoter fused to lacZ. The activity of chromosomally encoded FNR (cFNR) is shown for comparison. Cultures were grown in L broth under aerobic (filled bars) or anaerobic (open bars) conditions. The mean β-galactosidase activities in Miller units (MU) and standard deviations are shown (n = 9). The line indicates the amount of aerobic activity associated with wild-type FNR overproduced from pGS24.

Fig. 2.

Spectroscopic properties of reconstituted anaerobic FNR-S24F. (A) UV-visible spectra of 21.9 μM FNR-S24F cluster during a titration with O₂ as air-saturated buffer. The upper spectrum, with absorbance maxima at 320 and 405 nm, is the anaerobic sample before the addition of O₂ and is indicative of the presence of a [4Fe-4S] cluster. Subsequent spectra show changes upon the addition of 5, 10, 15, 29, and 51 μM O₂. After each O₂ addition, the incubations were continued for 90 min before collecting the absorbance spectra to ensure that the reactions were complete. Arrows show direction of absorbance change. The final spectrum, with absorbance maxima at 320, 420, and 500–600 nm, is characteristic of a [2Fe-2S] cluster. (Inset) Relationship between the O₂:[4Fe-4S]²⁺ ratio and the change in absorbance at 420 nm for FNR (gray line) and FNR-S24F (black line). (B) Comparison of the CD spectra of FNR (gray line) with FNR-S24F (black line).

Fig. 3.

The O₂ dependence and rate of the FNR-S24F [4Fe-4S]²⁺ cluster conversion upon addition of O₂. (A) Reaction of ≈20 μM FNR-S24F with different concentrations of O₂ (0, 20, 40, 60, 80, 100, and 150 μM) measured by optical absorbance at 420 nm. Data are shown in gray, and double-exponential function fits are plotted as black lines. Data are the average of at least three experiments. (B) First observed (pseudo-first-order) rate constants, from A, as a function of O₂ concentration. (C) Changes in EPR spectra of 20.4 μM FNR-S24F cluster during reaction with 200 μM O₂. The reaction was performed in a sealed anaerobic cuvette and initiated by the injection of air-saturated buffer. Samples were transferred to EPR tubes and immediately frozen. EPR parameters: temperature, 15 K; microwave power, 2.0 mW; frequency, 9.7 GHz; modulation, 5 G. (D) Signal intensity of the [3Fe-4S]⁺ intermediate (expressed as a percentage of the original [4Fe-4S]²⁺ cluster concentration) as a function of time for FNR (gray bars) and the FNR-S24F mutant protein (black bars).

Fig. 4.

Activity of E. coli FNR proteins with substitutions at residue 24. Plasmid pGS24 was subjected to QuikChange site-directed mutagenesis (Stratagene), and the plasmids expressing the indicated FNR variants were introduced to JRG1728 (fnr lac) containing pFF-41.5, a plasmid with an FNR-activated promoter fused to lacZ. Cultures were grown in L broth either aerobically (filled bars) or anaerobically (open bars). The mean β-galactosidase activities in MU ± SD are shown (n = 9). The horizontal lines indicate the amount of aerobic activity associated with overproduced wild-type FNR (Ser-24) and FNR-S24F.

Fig. 5.

Model of the reaction of FNR with oxygen and the importance of Ser-24. (A) Simple model of the FNR switch. FNR exists in two functional states: on, corresponding to the DNA-binding form (i.e., [4Fe-4S] FNR), and off, corresponding to the non-DNA-binding forms (i.e., [3Fe-4S], [2Fe-2S], and apo forms). The concentration of FNR in the cell is ≈6 μM and is relatively constant in the presence and absence of O₂ (21). An FNR-dependent reporter gene suggests that chromosomally encoded FNR is 2.5% active under fully aerobic conditions (Fig. 1, columns labeled cFNR), and thus the concentration of FNR in the off state under these conditions is ≈5.85 μM. The on to off switch (k₁ ≈270 M⁻¹ s⁻¹) is O₂-dependent. At a fully aerobic steady state (≈220 μM O₂), [on] = k₂/k₁ [off], or (0.15 × 10⁻⁶ = k₂/270 × 5.85 × 10⁻⁶), hence k₂ = ≈7 M⁻¹ s⁻¹. The switch from off to on (k₂) requires the incorporation of a [4Fe-4S] cluster, which depends on the supply of iron–sulfur clusters (FeS) from the biosynthetic machinery. Time-resolved transcriptomic studies suggest that the maximum FNR response during the switch from aerobic to anoxic growth occurs within 5–10 min (30), suggesting that the rate of off to on (v_ON) is ≈10 × 10⁻⁹ M s⁻¹. This information can be used to calculate a nominal concentration of FeS available for incorporation into FNR, v_ON = k₂ [off] [FeS], giving [FeS] = ≈240 μM. The model can be used to calculate FNR activity at different environmental O₂ concentrations, i.e., 50% FNR activity at ≈6 μM O₂ and 95% activity at ≈0.3 μM O₂. The values in parentheses are those specifically for FNR-S24F. (B) Alignment of amino acid sequences near the N-terminal cysteine residues of E. coli FNR and three FNR proteins from P. putida (ANR, PP_3287 and PP_3233). *, conserved residues; :, similar residues. The Arg (R) residue proposed to alter the O₂ sensitivity of PP_3387 relative to FNR/ANR is indicated in bold.

Fig. 6.

Model of the [4Fe-4S] binding region of FNR and FNR-S24F. (A) Alignment of amino acid sequences of the modeled region from FNR, endonuclease III, and thymine-DNA glycosylase. Bold type denotes the central Cys motif, connecting lines indicate residue identities, and numbers are the residue number in the full primary sequence of the respective molecules. (B and C) Predicted structure of FNR (B) and FNR-S24F (C) cluster regions produced in SWISS-Model by using the structure of endonuclease III (PDB code 2abk) as a template. Also represented is the sulfur atom of Cys-122 that binds the 4th iron atom of the iron-sulfur cluster. Images were produced by using PyMOL and show identical views. Note that the cluster ligand Cys-23 that is exposed in the FNR model (B) is shielded in the FNR-S24F model (C).