Mechanistic Insight into the Nitrosylation of the [4Fe−4S] Cluster of WhiB-like Proteins

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 104-fold faster than that observed with O2 and is by far the most rapid iron−sulfur cluster nitrosylation reaction reported to date. An overall stoichiometry of [Fe4S4(Cys)4]2− + 8NO → 2[FeI2(NO)4(Cys)2]0 + S2− + 3S0 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 [FeI4(NO)8(Cys)4]0 derived by dimerization of a pair of Roussin’s red ester (RRE) complexes.

Mechanism of [4Fe-4S](Cys)4 Cluster Nitrosylation Is Conserved among NO-responsive Regulators

“…Here we report detailed kinetic and thermodynamic investigations that reveal a rapid, multistep reaction of NO with the [4Fe-4S] cluster of FNR that, in terms of both rate and mechanism, is remarkably similar to that previously observed for Streptomyces and Mycobacterium Wbl proteins (11). These results suggest a common mechanism for the nitrosylation of the tetra-Cys ligated iron-sulfur clusters of different regulator proteins.…”

“…Until recently, little was known about the reactions of iron-sulfur clusters with NO in regulatory proteins. Wbl family proteins, found exclusively in the actinomycetes, contain a [4Fe-4S] cluster that undergoes a remarkably rapid and complex multistep reaction involving a total of eight NO molecules per cluster (11). The products of the reaction have the stoichiometry of [Fe(I) 2 (NO) 4 (SR) 2 ], similar to RRE 2 complexes.…”

Biochemical characterization and essentiality of fumarate hydratase

“…The UV-visible spectrum with absorption maxima at 360 and 405 nm indicates the presence of 4Fe-4S cluster in the enzyme and 78 % reconstitution efficiency using an Ɛ value of 16,000 M -1 cm -1 at 410 nm (30). The addition of sodium dithionite lowered the absorption intensity at 405 nm indicating a reduction of the cluster (Fig 3b) (31)(32)(33)(34) (21) and Trypanosoma cruzi (22) while for those from bacteria and archaea, the values are in the micromolar range. The catalytic efficiency (k cat /K m ) of PfFH∆40 is similar to L. major FH but 10-100 fold lower than that reported for other class I FHs ( Table 2).…”

The actinobacteria-specific gene wblA controls major developmental transitions in Streptomyces coelicolor A3(2)

“…Strikingly, binding of WhiB1 to a specific DNA target, resulting in repression of transcription in vitro, required interaction of the iron-sulphur cluster with nitric oxide (NO), an interaction that is specific and very rapid (Smith et al, 2010): WhiD of S. coelicolor also interacts strongly with NO (Crack et al, 2011). This suggests that Wbl proteins in general may change their regulatory properties in response to NO generated either exogenously (e.g.…”