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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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Stopped-flow kinetics of [4Fe−4S] WhiD nitrosylation. Stopped-flow kinetic traces following absorbance at (A) 360 nm and (B) 420 nm were recorded with WhiD (7.0 μM [4Fe−4S]) in the presence of 247 μM NO, giving a [NO]/[4Fe−4S] ratio of ∼35. (C) Stopped-flow kinetics traces following fluorescence changes upon mixing WhiD (2.0 μM [4Fe−4S]) with 145 μM NO, [NO]/[4Fe−4S] ratio of ∼72. Fits to each of the observed phases are drawn in (black lines). Insets show early events in the reaction time course.
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fig2: Stopped-flow kinetics of [4Fe−4S] WhiD nitrosylation. Stopped-flow kinetic traces following absorbance at (A) 360 nm and (B) 420 nm were recorded with WhiD (7.0 μM [4Fe−4S]) in the presence of 247 μM NO, giving a [NO]/[4Fe−4S] ratio of ∼35. (C) Stopped-flow kinetics traces following fluorescence changes upon mixing WhiD (2.0 μM [4Fe−4S]) with 145 μM NO, [NO]/[4Fe−4S] ratio of ∼72. Fits to each of the observed phases are drawn in (black lines). Insets show early events in the reaction time course.

Mentions: The kinetics of the reaction of [4Fe−4S] WhiD with NO in excess were measured using stopped-flow by monitoring A360 nm and A420 nm at, or close to, the maxima of nitrosylated product and the iron−sulfur cluster reactant, respectively, and by measurement of tryptophan fluorescence (Figure 2). Three distinct kinetic phases were observed at both wavelengths in absorbance, and by fluorescence, suggesting a three-step reaction, A → B → C → D. The data were fitted separately, and together, to exponential functions (Figure 2A, B, and D), giving equivalent results. Each absorbance data set gave similar apparent rate constants for the initial, rapid phase and the final, slower phase of the NO reaction. Plots of the observed pseudofirst-order rate constants (kobs) against NO concentration were linear for each step, indicating a first-order dependence on NO (see Figure 3A−D). However, the intermediate phase had quite different kinetic characteristics at 360 nm as compared to 420 nm, indicating that these two wavelengths report different processes in the intermediate part of the reaction. Therefore, the overall reaction should be modeled as a four-step reaction, that is, A → B → C → D → E, where the initial and final steps A → B and D → E, respectively, are detected at both wavelengths, while step B → C is detected at 360 nm and step C → D at 420 nm. The fluorescence response indicated that species B and C have similar quenching properties, resulting in a lag in the fluorescence recovery, and that D and E have similar fluorescence properties such that the final step, D → E, was not detected by fluorescence, consistent with the fluorescence titration data (Figure 1). The kinetic data are summarized in Table 1.


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)

Stopped-flow kinetics of [4Fe−4S] WhiD nitrosylation. Stopped-flow kinetic traces following absorbance at (A) 360 nm and (B) 420 nm were recorded with WhiD (7.0 μM [4Fe−4S]) in the presence of 247 μM NO, giving a [NO]/[4Fe−4S] ratio of ∼35. (C) Stopped-flow kinetics traces following fluorescence changes upon mixing WhiD (2.0 μM [4Fe−4S]) with 145 μM NO, [NO]/[4Fe−4S] ratio of ∼72. Fits to each of the observed phases are drawn in (black lines). Insets show early events in the reaction time course.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Stopped-flow kinetics of [4Fe−4S] WhiD nitrosylation. Stopped-flow kinetic traces following absorbance at (A) 360 nm and (B) 420 nm were recorded with WhiD (7.0 μM [4Fe−4S]) in the presence of 247 μM NO, giving a [NO]/[4Fe−4S] ratio of ∼35. (C) Stopped-flow kinetics traces following fluorescence changes upon mixing WhiD (2.0 μM [4Fe−4S]) with 145 μM NO, [NO]/[4Fe−4S] ratio of ∼72. Fits to each of the observed phases are drawn in (black lines). Insets show early events in the reaction time course.
Mentions: The kinetics of the reaction of [4Fe−4S] WhiD with NO in excess were measured using stopped-flow by monitoring A360 nm and A420 nm at, or close to, the maxima of nitrosylated product and the iron−sulfur cluster reactant, respectively, and by measurement of tryptophan fluorescence (Figure 2). Three distinct kinetic phases were observed at both wavelengths in absorbance, and by fluorescence, suggesting a three-step reaction, A → B → C → D. The data were fitted separately, and together, to exponential functions (Figure 2A, B, and D), giving equivalent results. Each absorbance data set gave similar apparent rate constants for the initial, rapid phase and the final, slower phase of the NO reaction. Plots of the observed pseudofirst-order rate constants (kobs) against NO concentration were linear for each step, indicating a first-order dependence on NO (see Figure 3A−D). However, the intermediate phase had quite different kinetic characteristics at 360 nm as compared to 420 nm, indicating that these two wavelengths report different processes in the intermediate part of the reaction. Therefore, the overall reaction should be modeled as a four-step reaction, that is, A → B → C → D → E, where the initial and final steps A → B and D → E, respectively, are detected at both wavelengths, while step B → C is detected at 360 nm and step C → D at 420 nm. The fluorescence response indicated that species B and C have similar quenching properties, resulting in a lag in the fluorescence recovery, and that D and E have similar fluorescence properties such that the final step, D → E, was not detected by fluorescence, consistent with the fluorescence titration data (Figure 1). The kinetic data are summarized in Table 1.

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