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. 2011 Feb 2;133(4):1112-21.
doi: 10.1021/ja109581t. Epub 2010 Dec 23.

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

Jason C Crack  1 , Laura J Smith, Melanie R Stapleton, Jamie Peck, Nicholas J Watmough, Mark J Buttner, Roger S Buxton, Jeffrey Green, Vasily S Oganesyan, Andrew J Thomson, Nick E Le Brun
Affiliations
Free PMC article

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

Jason C Crack et al. J Am Chem Soc. .
Free PMC article

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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Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
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.
Figure 3
Figure 3
Kinetic dependencies of the nitrosylation reaction on NO concentration. (A−D) Plots of the observed (pseudofirst order) rate constants (kobs) obtained from fits of the kinetic data in Figure 2 at 360 and 420 nm, over a range of NO concentrations. Panels A−D correspond to steps 1−4, respectively. kobs corresponds to A362 nm (light gray ●), A420 nm (dark gray ◼), and fluorescence (▲). Least-squares linear fits of the data are shown (black line), the gradient of which corresponds to the apparent second-order rate constant.
Figure 4
Figure 4
Stopped-flow kinetics of [4Fe−4S] M. tuberculosis WhiB1 nitrosylation. Stopped-flow kinetic traces following absorbance at (A) 364 nm and (B) 420 nm were recorded with WhiB1 (7.0 μM [4Fe−4S]) in the presence of 643 μM NO, giving a [NO]/[4Fe−4S] ratio of ∼92. Fits to each of the observed phases are drawn in (black lines). Insets show early events in the reaction time course. (C−F) Multiphase kinetics was observed for the reaction of [4Fe−4S] WhiB1 with NO over a range of concentrations (under pseudofirst-order conditions) at both 364 and 420 nm and observed rate constants (kobs) plotted as a function of NO concentration. Panels C−F corresponds to steps 1−4, respectively, of the four-step mechanism described in the text. kobs corresponds to A364 nm (●) and A420 nm (◼). Least-squares linear fits of the data are shown (black line), the gradient of which corresponds to the apparent second-order rate constant. Data for S. coelicolor WhiD, from Figure 3, are plotted for comparison (○). Overall, the data for WhiB1 were very similar to those for WhiD (see Table 1).
Figure 5
Figure 5
Quantification of sulfide release during WhiD cluster nitrosylation. (A) UV−visible absorbance spectra of 12.6 μM WhiD containing 7.9 μM [4Fe−4S] in assay buffer pH 8.0 before (solid black line) and after (solid olive green line) the addition of DTNB (192 μM final concentration). The arrow indicates ΔA420 nm, which results from the free thiol content of the sample. (B) The sample in (A) (dashed black and olive lines) following the addition of NO, as PROLI-NONOate (final NO concentration 141.5 μM) (solid olive line). The spectrum in solid black resulted from an equivalent addition of NO, as PROLI-NONOate, to an identical sample in the absence of DTNB. From these two spectra, the ΔA420 nm due to sulfide released during cluster nitrosylation was calculated. A correction factor was applied to account for the slow loss (∼7%) of TNB anions during the course of the assay.(43) In the assay buffer at pH 8.0, TNB anions displayed a slightly shifted absorbance maximum at 420 nm, with ε420 nm = 13 200 M−1 cm−1. All absorbance changes correspond to sample 3 in Table 4.
Figure 6
Figure 6
Roussin’s red ester (RRE) and a proposed novel octa-nitrosyl cluster resulting from nitrosylation of the [4Fe−4S] cluster of Wbl proteins. DFT energy-minimized structures of RRE, [FeI2(NO)4(CH3S−)2]0, with bridging methyl thiolates in the trans (A) and cis (B) conformations. Calculations revealed that, in vacuo, the cis and trans configurations have identical relative energies. Bond lengths (in Å) are indicated. (C) DFT energy-minimized structure of a pair RREs species, with bridging methyl thiolates, [FeI4(NO)8(SCH3)4]0, having an overall point symmetry of D2d with the inter-RRE direction aligned along the S4 axis. The Fe(NO)2 unit lies in a plane that minimizes the steric interaction with the tricapping thiol groups. The Fe4 unit is flattened from the regular Td shape along the S4 axis. This is apparent from the Fe−S distance of 2.37 Å that links the RREs as compared to a Fe−S distance of 2.54 Å within a RRE. Thus, the Fe−Fe distance across the rhomb increased from 2.63 to 3.02 Å, and the S−S distance increased from 3.63 to 4.07 Å; see Table S1 for full details of interatomic distances.
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