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Influence of association state and DNA binding on the O₂-reactivity of [4Fe-4S] fumarate and nitrate reduction (FNR) regulator.

Crack JC, Stapleton MR, Green J, Thomson AJ, Le Brun NE - Biochem. J. (2014)

Bottom Line: In vivo studies of permanently dimeric D154A FNR show that DNA binding does not affect the rate of cluster incorporation into the apoprotein or the rate of O2-mediated cluster loss.Decoupling leads to an increase in the rate of the [3Fe-4S]1+ into [2Fe-2S]2+ conversion step, consistent with the suggestion that this step drives association state changes in the wild-type protein.We have also shown that DNA-bound FNR reacts more rapidly with O2 than FNR free in solution, implying that transcriptionally active FNR is the preferred target for reaction with O2.

View Article: PubMed Central - PubMed

Affiliation: *Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, U.K.

ABSTRACT
The fumarate and nitrate reduction (FNR) regulator is the master switch for the transition between anaerobic and aerobic respiration in Escherichia coli. Reaction of dimeric [4Fe-4S] FNR with O2 results in conversion of the cluster into a [2Fe-2S] form, via a [3Fe-4S] intermediate, leading to the loss of DNA binding through dissociation of the dimer into monomers. In the present paper, we report studies of two previously identified variants of FNR, D154A and I151A, in which the form of the cluster is decoupled from the association state. In vivo studies of permanently dimeric D154A FNR show that DNA binding does not affect the rate of cluster incorporation into the apoprotein or the rate of O2-mediated cluster loss. In vitro studies show that O2-mediated cluster conversion for D154A and the permanent monomer I151A FNR is the same as in wild-type FNR, but with altered kinetics. Decoupling leads to an increase in the rate of the [3Fe-4S]1+ into [2Fe-2S]2+ conversion step, consistent with the suggestion that this step drives association state changes in the wild-type protein. We have also shown that DNA-bound FNR reacts more rapidly with O2 than FNR free in solution, implying that transcriptionally active FNR is the preferred target for reaction with O2.

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Kinetics of wild-type FNR cluster conversion in the presence of DNAThe rate of O2-dependent cluster conversion of (A) wild-type FNR (~1 μM [4Fe-4S] FNR, equivalent to ~0.5 μM dimer); (B) as (A) but in the presence of pGS422 (~0.6 μM); (C) as (A) but in the presence of a 345-bp FF-41.5 DNA fragment (~0.6 μM) was measured by absorbance at 406 nm. Reactions at two O2 concentrations are shown for each condition. Data (shown in grey) are averages of three measurements; fits to the experimental data are in black. (D) Plots of the first observed (first-order) rate constants obtained from the data in (A–C) and additional experiments for FNR (white circles), FNR in the presence of pGS422 (black circles), and FNR in the presence of a 345-bp FF-41.5 fragment (grey squares) as a function of the O2 concentration. Least squares linear fits of the data are drawn in for FNR and FNR in the presence of pGS422, the gradients of which correspond to the apparent second-order rate constants. The buffer was 20 mM Tris/HCl and 5% glycerol, pH 8.0.
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Figure 7: Kinetics of wild-type FNR cluster conversion in the presence of DNAThe rate of O2-dependent cluster conversion of (A) wild-type FNR (~1 μM [4Fe-4S] FNR, equivalent to ~0.5 μM dimer); (B) as (A) but in the presence of pGS422 (~0.6 μM); (C) as (A) but in the presence of a 345-bp FF-41.5 DNA fragment (~0.6 μM) was measured by absorbance at 406 nm. Reactions at two O2 concentrations are shown for each condition. Data (shown in grey) are averages of three measurements; fits to the experimental data are in black. (D) Plots of the first observed (first-order) rate constants obtained from the data in (A–C) and additional experiments for FNR (white circles), FNR in the presence of pGS422 (black circles), and FNR in the presence of a 345-bp FF-41.5 fragment (grey squares) as a function of the O2 concentration. Least squares linear fits of the data are drawn in for FNR and FNR in the presence of pGS422, the gradients of which correspond to the apparent second-order rate constants. The buffer was 20 mM Tris/HCl and 5% glycerol, pH 8.0.

Mentions: To investigate the effects of DNA on the kinetics of cluster conversion, anaerobic wild-type [4Fe-4S] FNR was mixed with aliquots of pGS422, a plasmid containing a consensus (TTGATGTACATCAA) FNR-binding site, in buffer C (see the Materials and methods section), to give a 1.2-fold excess of DNA. Wild-type FNR was shown previously to bind specifically (Kd ~14 nM) to the FF-41.5 promoter in anaerobic buffer in an O2-dependent manner [34,35]. In the absence of pGS422, cluster conversion occurred in an O2-dependent manner under pseudo-first-order conditions, as observed previously [14]. A double exponential function was needed to fit the data. The second phase of the reaction, which only begins to contribute significantly towards the end of 100 s acquisition period, corresponds to a slow increase in absorbance; this is unusual, but has been observed previously under certain conditions [13] and is believed to be associated with the instability of the [3Fe-4S]1+ intermediate, or with the propensity of ejected Fe2+ to precipitate. Despite this, the rate constant for the initial step can be readily obtained [13]; the apparent second-order rate constant under these conditions (in buffer C) was k1=229 (±10) M−1·s−1 (Figures 7A and 7D). In the presence of pGS422, the datasets were best described by a single exponential function (Figure 7B), indicating that the conversion of the [3Fe-4S]1+ intermediate into the [2Fe-2S] form occurs more rapidly, such that the two steps of cluster conversion cannot be easily distinguished when FNR is DNA-bound. Similar observations were made previously for FNR in the presence of a Fe3+ chelator [13]. Plotting kobs obtained in the presence of pGS422 against the O2 concentration revealed a linear dependence on O2, with an apparent second-order rate constant, k1=439 (±28) M−1·s−1, approximately twice that of FNR in the absence of pGS422 (Figure 7D). Experiments using a 345-bp FF-41.5 linear DNA fragment in place of pGS422 gave results very similar to those obtained with supercoiled plasmid DNA (Figures 7C and 7D).


Influence of association state and DNA binding on the O₂-reactivity of [4Fe-4S] fumarate and nitrate reduction (FNR) regulator.

Crack JC, Stapleton MR, Green J, Thomson AJ, Le Brun NE - Biochem. J. (2014)

Kinetics of wild-type FNR cluster conversion in the presence of DNAThe rate of O2-dependent cluster conversion of (A) wild-type FNR (~1 μM [4Fe-4S] FNR, equivalent to ~0.5 μM dimer); (B) as (A) but in the presence of pGS422 (~0.6 μM); (C) as (A) but in the presence of a 345-bp FF-41.5 DNA fragment (~0.6 μM) was measured by absorbance at 406 nm. Reactions at two O2 concentrations are shown for each condition. Data (shown in grey) are averages of three measurements; fits to the experimental data are in black. (D) Plots of the first observed (first-order) rate constants obtained from the data in (A–C) and additional experiments for FNR (white circles), FNR in the presence of pGS422 (black circles), and FNR in the presence of a 345-bp FF-41.5 fragment (grey squares) as a function of the O2 concentration. Least squares linear fits of the data are drawn in for FNR and FNR in the presence of pGS422, the gradients of which correspond to the apparent second-order rate constants. The buffer was 20 mM Tris/HCl and 5% glycerol, pH 8.0.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214427&req=5

Figure 7: Kinetics of wild-type FNR cluster conversion in the presence of DNAThe rate of O2-dependent cluster conversion of (A) wild-type FNR (~1 μM [4Fe-4S] FNR, equivalent to ~0.5 μM dimer); (B) as (A) but in the presence of pGS422 (~0.6 μM); (C) as (A) but in the presence of a 345-bp FF-41.5 DNA fragment (~0.6 μM) was measured by absorbance at 406 nm. Reactions at two O2 concentrations are shown for each condition. Data (shown in grey) are averages of three measurements; fits to the experimental data are in black. (D) Plots of the first observed (first-order) rate constants obtained from the data in (A–C) and additional experiments for FNR (white circles), FNR in the presence of pGS422 (black circles), and FNR in the presence of a 345-bp FF-41.5 fragment (grey squares) as a function of the O2 concentration. Least squares linear fits of the data are drawn in for FNR and FNR in the presence of pGS422, the gradients of which correspond to the apparent second-order rate constants. The buffer was 20 mM Tris/HCl and 5% glycerol, pH 8.0.
Mentions: To investigate the effects of DNA on the kinetics of cluster conversion, anaerobic wild-type [4Fe-4S] FNR was mixed with aliquots of pGS422, a plasmid containing a consensus (TTGATGTACATCAA) FNR-binding site, in buffer C (see the Materials and methods section), to give a 1.2-fold excess of DNA. Wild-type FNR was shown previously to bind specifically (Kd ~14 nM) to the FF-41.5 promoter in anaerobic buffer in an O2-dependent manner [34,35]. In the absence of pGS422, cluster conversion occurred in an O2-dependent manner under pseudo-first-order conditions, as observed previously [14]. A double exponential function was needed to fit the data. The second phase of the reaction, which only begins to contribute significantly towards the end of 100 s acquisition period, corresponds to a slow increase in absorbance; this is unusual, but has been observed previously under certain conditions [13] and is believed to be associated with the instability of the [3Fe-4S]1+ intermediate, or with the propensity of ejected Fe2+ to precipitate. Despite this, the rate constant for the initial step can be readily obtained [13]; the apparent second-order rate constant under these conditions (in buffer C) was k1=229 (±10) M−1·s−1 (Figures 7A and 7D). In the presence of pGS422, the datasets were best described by a single exponential function (Figure 7B), indicating that the conversion of the [3Fe-4S]1+ intermediate into the [2Fe-2S] form occurs more rapidly, such that the two steps of cluster conversion cannot be easily distinguished when FNR is DNA-bound. Similar observations were made previously for FNR in the presence of a Fe3+ chelator [13]. Plotting kobs obtained in the presence of pGS422 against the O2 concentration revealed a linear dependence on O2, with an apparent second-order rate constant, k1=439 (±28) M−1·s−1, approximately twice that of FNR in the absence of pGS422 (Figure 7D). Experiments using a 345-bp FF-41.5 linear DNA fragment in place of pGS422 gave results very similar to those obtained with supercoiled plasmid DNA (Figures 7C and 7D).

Bottom Line: In vivo studies of permanently dimeric D154A FNR show that DNA binding does not affect the rate of cluster incorporation into the apoprotein or the rate of O2-mediated cluster loss.Decoupling leads to an increase in the rate of the [3Fe-4S]1+ into [2Fe-2S]2+ conversion step, consistent with the suggestion that this step drives association state changes in the wild-type protein.We have also shown that DNA-bound FNR reacts more rapidly with O2 than FNR free in solution, implying that transcriptionally active FNR is the preferred target for reaction with O2.

View Article: PubMed Central - PubMed

Affiliation: *Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, U.K.

ABSTRACT
The fumarate and nitrate reduction (FNR) regulator is the master switch for the transition between anaerobic and aerobic respiration in Escherichia coli. Reaction of dimeric [4Fe-4S] FNR with O2 results in conversion of the cluster into a [2Fe-2S] form, via a [3Fe-4S] intermediate, leading to the loss of DNA binding through dissociation of the dimer into monomers. In the present paper, we report studies of two previously identified variants of FNR, D154A and I151A, in which the form of the cluster is decoupled from the association state. In vivo studies of permanently dimeric D154A FNR show that DNA binding does not affect the rate of cluster incorporation into the apoprotein or the rate of O2-mediated cluster loss. In vitro studies show that O2-mediated cluster conversion for D154A and the permanent monomer I151A FNR is the same as in wild-type FNR, but with altered kinetics. Decoupling leads to an increase in the rate of the [3Fe-4S]1+ into [2Fe-2S]2+ conversion step, consistent with the suggestion that this step drives association state changes in the wild-type protein. We have also shown that DNA-bound FNR reacts more rapidly with O2 than FNR free in solution, implying that transcriptionally active FNR is the preferred target for reaction with O2.

Show MeSH
Related in: MedlinePlus