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

Abstract

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.

Interaction of the Streptomyces Wbl protein WhiD with the principal sigma factor σHrdB depends on the WhiD [4Fe-4S] cluster

“…Interestingly, no nitrosylated forms of the WhiD [4Fe-4S] cluster, nor any iron-nitrosyl products were detected by non-denaturing mass spectrometry. Such species were recently detected by mass spectrometry for ScWhiD (22), suggesting that NO-complexes of SvWhiD are less stable under the conditions of the mass spectrometry experiments than those of ScWhiD.…”

Characterization of [2Fe–2S]‐Cluster‐Bridged Protein Complexes and Reaction Intermediates by use of Native Mass Spectrometric Methods

“…For example, Crack et al. first illustrated the application of nMS to study physiologically relevant cluster chemistry . However, use of the application has been limited due to challenges from cluster instability and from the additional sample preparation steps required to spray intact cluster‐bound proteins from mass spectrometric compatible buffers.…”

“…For example, Crack et al first illustrated the application of nMS to study physiologically relevant cluster chemistry. [12][13][14][15][16][17][18][19][20][21][22] However, use of the application has been limited due to challenges from cluster instability and from the additional sample preparation steps required to spray intact cluster-bound proteins from mass spectrometric compatible buffers. The majority of such proteins characterized by mass spectrometry have Fe-S clusters buried inside the protein core and not at the protein-protein interface.…”

Dioxygen controls the nitrosylation reactions of a protein-bound [4Fe4S] cluster

“…While the number of available thiols is unchanged between the anaerobic and aerobic nitrosylation of A. ferroxidans HiPIP, the O 2 could potentially facilitate elimination of the bridging sulfides. Oxidation of the bridging sulfides has been observed in the nitrosylation of other protein-bound [4Fe4S] clusters with the sulfides remaining bound to the protein as persulfide cysteine, 15,35 and indeed Crack et al observed formation of RRE products in the nitrosylation of several [4Fe4S] proteins. 7 We know that thiolate DNIC species can be converted to RRE under oxidising conditions with the thiolate sulfides as targets of oxidation and disulfide as a byproduct.…”

“…32 Non-denaturing mass spectrometry and NRVS have been used productively to identify further products of the nitrosylation reactions of [4Fe4S] proteins. 9,10,[14][15][16][33][34][35] We previously reported IR spectroscopy alongside other spectroscopic methods to show that [2Fe2S] Spinach ferredoxin (Fd) reacts with NO in a manner dependent on the redox environment of the reaction. Nitrosylation of this protein under anaerobic conditions yields a mixture of DNIC and RRE, whereas, in the presence of trace O 2 or the thiolate sequestering reagent, iodoacetamide, RRE is formed as the major product.…”