Mass Spectrometric Identification of [4Fe-4S](NO)x Intermediates of Nitric Oxide Sensing by Regulatory Iron-Sulfur Cluster Proteins.

Nitric oxide (NO) can function as both a cytotoxin and a signalling molecule. In both cases, reaction with iron-sulfur (Fe-S) cluster proteins plays an important role because Fe-S clusters are reactive towards NO and so are a primary site of general NO-induced damage (toxicity). This sensitivity to nitrosylation is harnessed in the growing group of regulatory proteins that function in sensing of NO via an Fe-S cluster. Although information about the products of cluster nitrosylation is now emerging, detection and identification of intermediates remains a major challenge, due to their transient nature and the difficulty in distinguishing spectroscopically similar iron-NO species. Here we report studies of the NO-sensing Fe-S cluster regulators NsrR and WhiD using non-denaturing mass spectrometry, in which non-covalent interactions between the protein and Fe/S/NO species are preserved. The data provide remarkable insight into the nitrosylation reactions, permitting identification, for the first time, of protein-bound mono-, di- and tetranitrosyl [4Fe-4S] cluster complexes ([4Fe-4S](NO), [4Fe-4S])(NO)2 and [4Fe-4S](NO)4 ) as intermediates along pathways to formation of product Roussin's red ester (RRE) and Roussin's black salt (RBS)-like species. The data allow the nitrosylation mechanisms of NsrR and WhiD to be elucidated and clearly distinguished.