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Mechanistic insight into the nitrosylation of the [4Fe-4S] cluster of WhiB-like proteins.

Crack JC, Smith LJ, Stapleton MR, Peck J, Watmough NJ, Buttner MJ, Buxton RS, Green J, Oganesyan VS, Thomson AJ, Le Brun NE - J. Am. Chem. Soc. (2010)

Bottom Line: The reaction is 10(4)-fold faster than that observed with O(2) and is by far the most rapid iron-sulfur cluster nitrosylation reaction reported to date.Kinetic analysis leads to a four-step mechanism that accounts for the observed NO dependence.DFT calculations suggest the possibility that the nitrosylation product is a novel cluster [Fe(I)(4)(NO)(8)(Cys)(4)](0) derived by dimerization of a pair of Roussin's red ester (RRE) complexes.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich NR4 7TJ, United Kingdom.

ABSTRACT
The reactivity of protein bound iron-sulfur clusters with nitric oxide (NO) is well documented, but little is known about the actual mechanism of cluster nitrosylation. Here, we report studies of members of the Wbl family of [4Fe-4S] containing proteins, which play key roles in regulating developmental processes in actinomycetes, including Streptomyces and Mycobacteria, and have been shown to be NO responsive. Streptomyces coelicolor WhiD and Mycobacterium tuberculosis WhiB1 react extremely rapidly with NO in a multiphasic reaction involving, remarkably, 8 NO molecules per [4Fe-4S] cluster. The reaction is 10(4)-fold faster than that observed with O(2) and is by far the most rapid iron-sulfur cluster nitrosylation reaction reported to date. An overall stoichiometry of [Fe(4)S(4)(Cys)(4)](2-) + 8NO → 2[Fe(I)(2)(NO)(4)(Cys)(2)](0) + S(2-) + 3S(0) has been established by determination of the sulfur products and their oxidation states. Kinetic analysis leads to a four-step mechanism that accounts for the observed NO dependence. DFT calculations suggest the possibility that the nitrosylation product is a novel cluster [Fe(I)(4)(NO)(8)(Cys)(4)](0) derived by dimerization of a pair of Roussin's red ester (RRE) complexes.

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Titration of [4Fe−4S] WhiD with NO. (A) Absorbance spectra of [4Fe−4S] WhiD (29 μM) following additions of NO giving [NO]/[WhiD] ratios up to 11.5. (B) CD spectra of a titration equivalent to that in (A). (C) Fluorescence spectra of WhiD (1.9 μM; excitation at 280 nm; excitation and emission slit widths of 3 and 4 nm, respectively) following additions of NO giving [NO]/[WhiD] ratios up to 10.4. (D) Changes in the optical spectra, ΔA362 nm (green ●), ΔCD324 nm (blue ●), and ΔFI354 nm (●), were normalized and plotted versus the [NO]:[4Fe−4S] cluster ratio. Tangents to the initial slope and the titration end points of the absorbance and CD data are drawn in. The buffer was 20 mM Tris, 20 mM Bis-Tris-propane, 20 mM MES, 100 mM NaCl, and 5% glycerol pH 8.0.
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fig1: Titration of [4Fe−4S] WhiD with NO. (A) Absorbance spectra of [4Fe−4S] WhiD (29 μM) following additions of NO giving [NO]/[WhiD] ratios up to 11.5. (B) CD spectra of a titration equivalent to that in (A). (C) Fluorescence spectra of WhiD (1.9 μM; excitation at 280 nm; excitation and emission slit widths of 3 and 4 nm, respectively) following additions of NO giving [NO]/[WhiD] ratios up to 10.4. (D) Changes in the optical spectra, ΔA362 nm (green ●), ΔCD324 nm (blue ●), and ΔFI354 nm (●), were normalized and plotted versus the [NO]:[4Fe−4S] cluster ratio. Tangents to the initial slope and the titration end points of the absorbance and CD data are drawn in. The buffer was 20 mM Tris, 20 mM Bis-Tris-propane, 20 mM MES, 100 mM NaCl, and 5% glycerol pH 8.0.

Mentions: Sequential additions of the NO releasing reagent PROLI-NONOate to an anaerobic sample of holo-WhiD were followed by UV−visible absorption and by near UV−visible CD spectroscopy; see Figure 1A and B. The progressive decrease in absorbance at 406 nm, the increase in the absorbance at 362 nm, and the lesser increase in the region 500−700 nm gave apparent isosbestic points at 398, 480, and 700 nm. A plot of ΔA362 nm against the ratio [NO]:[4Fe−4S] (Figure 1D) revealed that the reaction went to completion with a stoichiometry of 8.6 (±0.25) NO molecules per [4Fe−4S] cluster. CD signals arising from the cluster decreased almost to zero as the reaction with NO proceeded. A plot of the CD intensity at 324 nm against the ratio [NO]:[4Fe−4S] gave a similar reaction stoichiometry of 7.8 (±0.5) NO molecules per [4Fe−4S] cluster (Figure 1D). The final UV−visible spectrum (Figure 1A) was similar to that of an RRE,15,17 with a principal absorption band at 362 nm and a shoulder at ∼430 nm. Based on a model compound extinction coefficient (ε362 nm = 8530 M−1 cm−1,(17) the spectrum indicated the presence of two RREs per WhiD, consistent with the observed reaction stoichiometry. The WhiD NO reaction product was stable for many hours under anaerobic conditions, but was slowly lost in aerobic buffers.


Mechanistic insight into the nitrosylation of the [4Fe-4S] cluster of WhiB-like proteins.

Crack JC, Smith LJ, Stapleton MR, Peck J, Watmough NJ, Buttner MJ, Buxton RS, Green J, Oganesyan VS, Thomson AJ, Le Brun NE - J. Am. Chem. Soc. (2010)

Titration of [4Fe−4S] WhiD with NO. (A) Absorbance spectra of [4Fe−4S] WhiD (29 μM) following additions of NO giving [NO]/[WhiD] ratios up to 11.5. (B) CD spectra of a titration equivalent to that in (A). (C) Fluorescence spectra of WhiD (1.9 μM; excitation at 280 nm; excitation and emission slit widths of 3 and 4 nm, respectively) following additions of NO giving [NO]/[WhiD] ratios up to 10.4. (D) Changes in the optical spectra, ΔA362 nm (green ●), ΔCD324 nm (blue ●), and ΔFI354 nm (●), were normalized and plotted versus the [NO]:[4Fe−4S] cluster ratio. Tangents to the initial slope and the titration end points of the absorbance and CD data are drawn in. The buffer was 20 mM Tris, 20 mM Bis-Tris-propane, 20 mM MES, 100 mM NaCl, and 5% glycerol pH 8.0.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117330&req=5

fig1: Titration of [4Fe−4S] WhiD with NO. (A) Absorbance spectra of [4Fe−4S] WhiD (29 μM) following additions of NO giving [NO]/[WhiD] ratios up to 11.5. (B) CD spectra of a titration equivalent to that in (A). (C) Fluorescence spectra of WhiD (1.9 μM; excitation at 280 nm; excitation and emission slit widths of 3 and 4 nm, respectively) following additions of NO giving [NO]/[WhiD] ratios up to 10.4. (D) Changes in the optical spectra, ΔA362 nm (green ●), ΔCD324 nm (blue ●), and ΔFI354 nm (●), were normalized and plotted versus the [NO]:[4Fe−4S] cluster ratio. Tangents to the initial slope and the titration end points of the absorbance and CD data are drawn in. The buffer was 20 mM Tris, 20 mM Bis-Tris-propane, 20 mM MES, 100 mM NaCl, and 5% glycerol pH 8.0.
Mentions: Sequential additions of the NO releasing reagent PROLI-NONOate to an anaerobic sample of holo-WhiD were followed by UV−visible absorption and by near UV−visible CD spectroscopy; see Figure 1A and B. The progressive decrease in absorbance at 406 nm, the increase in the absorbance at 362 nm, and the lesser increase in the region 500−700 nm gave apparent isosbestic points at 398, 480, and 700 nm. A plot of ΔA362 nm against the ratio [NO]:[4Fe−4S] (Figure 1D) revealed that the reaction went to completion with a stoichiometry of 8.6 (±0.25) NO molecules per [4Fe−4S] cluster. CD signals arising from the cluster decreased almost to zero as the reaction with NO proceeded. A plot of the CD intensity at 324 nm against the ratio [NO]:[4Fe−4S] gave a similar reaction stoichiometry of 7.8 (±0.5) NO molecules per [4Fe−4S] cluster (Figure 1D). The final UV−visible spectrum (Figure 1A) was similar to that of an RRE,15,17 with a principal absorption band at 362 nm and a shoulder at ∼430 nm. Based on a model compound extinction coefficient (ε362 nm = 8530 M−1 cm−1,(17) the spectrum indicated the presence of two RREs per WhiD, consistent with the observed reaction stoichiometry. The WhiD NO reaction product was stable for many hours under anaerobic conditions, but was slowly lost in aerobic buffers.

Bottom Line: The reaction is 10(4)-fold faster than that observed with O(2) and is by far the most rapid iron-sulfur cluster nitrosylation reaction reported to date.Kinetic analysis leads to a four-step mechanism that accounts for the observed NO dependence.DFT calculations suggest the possibility that the nitrosylation product is a novel cluster [Fe(I)(4)(NO)(8)(Cys)(4)](0) derived by dimerization of a pair of Roussin's red ester (RRE) complexes.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich NR4 7TJ, United Kingdom.

ABSTRACT
The reactivity of protein bound iron-sulfur clusters with nitric oxide (NO) is well documented, but little is known about the actual mechanism of cluster nitrosylation. Here, we report studies of members of the Wbl family of [4Fe-4S] containing proteins, which play key roles in regulating developmental processes in actinomycetes, including Streptomyces and Mycobacteria, and have been shown to be NO responsive. Streptomyces coelicolor WhiD and Mycobacterium tuberculosis WhiB1 react extremely rapidly with NO in a multiphasic reaction involving, remarkably, 8 NO molecules per [4Fe-4S] cluster. The reaction is 10(4)-fold faster than that observed with O(2) and is by far the most rapid iron-sulfur cluster nitrosylation reaction reported to date. An overall stoichiometry of [Fe(4)S(4)(Cys)(4)](2-) + 8NO → 2[Fe(I)(2)(NO)(4)(Cys)(2)](0) + S(2-) + 3S(0) has been established by determination of the sulfur products and their oxidation states. Kinetic analysis leads to a four-step mechanism that accounts for the observed NO dependence. DFT calculations suggest the possibility that the nitrosylation product is a novel cluster [Fe(I)(4)(NO)(8)(Cys)(4)](0) derived by dimerization of a pair of Roussin's red ester (RRE) complexes.

Show MeSH
Related in: MedlinePlus