Nitrosylation of Nitric‐Oxide‐Sensing Regulatory Proteins Containing [4Fe‐4S] Clusters Gives Rise to Multiple Iron–Nitrosyl Complexes

Abstract

The reaction of protein-bound iron-sulfur (Fe-S) clusters with nitric oxide (NO) playskey roles in NO-mediated toxicity and signaling. Elucidation of the mechanism of the reaction of NO with DNAregulatory proteins that contain Fe-S clusters has been hampered by alack of information about the nature of the iron-nitrosyl products formed. Herein, we report nuclear resonance vibrational spectroscopy( NRVS) and density functional theory (DFT) calculations that identify NO reaction products in WhiD and NsrR, regulatory proteins that use a[4Fe-4S] cluster to sense NO.This work reveals that nitrosylation yields multiple products structurally related to RoussinsR ed Ester (RRE, [Fe 2 (NO) 4 (Cys) 2 ]) and Roussins BlackS alt (RBS,[ Fe 4 (NO) 7 S 3 ]. In the latter case,t he absence of 32 S/ 34 Sshifts in the FeÀSregion of the NRVS spectra suggest that an ew species,R oussinsB lack Ester (RBE), may be formed, in which one or more of the sulfide ligands is replaced by Cys thiolates.

Crystal structures of the NO sensor NsrR reveal how its iron-sulfur cluster modulates DNA binding

“…In all cases nitrosylation resulted in loss of the 412 nm band and emergence of a new band at 357 nm, as previously reported for wild type ScNsrR8, indicating similarity in the final iron nitrosyl products formed7 (Supplementary Fig. 10a and b).…”

13. Reactivity of iron-sulfur clusters with nitric oxide

“…From the NRVS, there was a smaller component of RRE compared to in the NsrR spectrum, and significantly less DNIC present. This is consistent with EPR studies that showed that DNIC accounted for only ~3% of the total initial cluster iron [41,47].…”

“…This species is predicted to have an NRVS spectrum similar to RBS, but importantly with small or no shifts due to the 32 S/ 34 S isotope substitution. DFT calculations also indicated that Cys persulfide forms of these iron-nitrosyls could be present because their calculated NRVS spectra are not significantly different from the Cys thiolate forms [47].…”

“…In vivo many transcriptional regulators respond to the presence of aqueous NO, acidified nitrite or S-nitroso-glutathione; in E.coli these include MetR, IscR, Fur, FNR, SoxR, OxyR, NorR and NsrR (formally yjeB). In E. coli, NsrR was shown to sense NO and modulate a regulon of at least 60 genes, including nitrate (Nar) and nitrite reductase (Nir), as well as hcp and hmp, which encode NO detoxifying enzymes [47,109,114,118,119].…”

Redox-Sensing Iron–Sulfur Cluster Regulators

“…In vivo, many transcriptional regulators respond to the presence of aqueous NO, acidified nitrite or S-nitroso-glutathione; in E. coli these include MetR, IscR, Fur, FNR, SoxR, OxyR, NorR and NsrR. This can lead to a dramatic metabolic reorganization in order to adapt to stress conditions (46,48,99,132,146 Figure 7). This species has invariably been detected, both in vivo These studies revealed a rapid multi-NO (~8 per cluster), multi-phasic reaction that appears to be characteristic of [4Fe-4S] cluster containing regulators.…”