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

Redox-Sensing Iron–Sulfur Cluster Regulators

Significance: Iron–sulfur cluster proteins carry out multiple functions, including as regulators of gene transcription/translation in response to environmental stimuli. In all known cases, the cluster acts as the sensory module, where the inherent reactivity/fragility of iron–sulfur clusters with small/redox-active molecules is exploited to effect conformational changes that modulate binding to DNA regulatory sequences. This promotes an often substantial reprogramming of the cellular proteome that enables the organism or cell to adapt to, or counteract, its changing circumstances. Recent Advances: Significant progress has been made recently in the structural and mechanistic characterization of iron–sulfur cluster regulators and, in particular, the O2 and NO sensor FNR, the NO sensor NsrR, and WhiB-like proteins of Actinobacteria. These are the main focus of this review. Critical Issues: Striking examples of how the local environment controls the cluster sensitivity and reactivity are now emerging, but the basis for this is not yet fully understood for any regulatory family. Future Directions: Characterization of iron–sulfur cluster regulators has long been hampered by a lack of high-resolution structural data. Although this still presents a major future challenge, recent advances now provide a firm foundation for detailed understanding of how a signal is transduced to effect gene regulation. This requires the identification of often unstable intermediate species, which are difficult to detect and may be hard to distinguish using traditional techniques. Novel approaches will be required to solve these problems

Influence of association state and DNA binding on the O2-reactivity of [4Fe-4S] fumarate and nitrate reduction (FNR) regulator

The fumarate and nitrate reduction (FNR) regulator is the master switch for the transition between anaerobic and aerobic respiration in Escherichia coli. Reaction of dimeric [4Fe-4S] FNR with O2 results in conversion of the cluster into a [2Fe-2S] form, via a [3Fe-4S] intermediate, leading to the loss of DNA binding through dissociation of the dimer into monomers. In the present paper, we report studies of two previously identified variants of FNR, D154A and I151A, in which the form of the cluster is decoupled from the association state. In vivo studies of permanently dimeric D154A FNR show that DNA binding does not affect the rate of cluster incorporation into the apoprotein or the rate of O2-mediated cluster loss. In vitro studies show that O2-mediated cluster conversion for D154A and the permanent monomer I151A FNR is the same as in wild-type FNR, but with altered kinetics. Decoupling leads to an increase in the rate of the [3Fe-4S]1+ into [2Fe-2S]2+ conversion step, consistent with the suggestion that this step drives association state changes in the wild-type protein. We have also shown that DNA-bound FNR reacts more rapidly with O2 than FNR free in solution, implying that transcriptionally active FNR is the preferred target for reaction with O2

Mass spectrometric detection of iron nitrosyls, sulfide oxidation and mycothiolation during nitrosylation of the NO sensor [4Fe-4S] NsrR

Identification of RRE-type iron-nitrosyl species formed upon nitrosylation of [4Fe–4S] NsrR.</p

Mass spectrometric identification of intermediates in the O2-driven [4Fe-4S] to [2Fe-2S] cluster conversion in FNR

The iron-sulfur cluster containing protein FNR is the master regulator for the switch between anaerobic and aerobic respiration in Escherichia coli and many other bacteria. The [4Fe-4S] cluster functions as the sensory module, undergoing reaction with O2 that leads to conversion to a [2Fe-2S] form with loss of high affinity DNA-binding. Here we report studies of the FNR cluster conversion reaction using time-resolved electrospray ionization mass spectrometry. The data provide new insight into the reaction, permitting the detection of cluster conversion intermediates and products, including a novel [3Fe-3S] cluster and persulfide coordinated [2Fe-2S] clusters ([2Fe-2S](S)n, where n = 1 or 2). Analysis of kinetic data revealed a branched mechanism in which cluster sulfide oxidation occurs in parallel with cluster conversion, and not as a subsequent, secondary reaction, to generate ([2Fe-2S](S)n species. This methodology shows great potential for broad application to studies of protein cofactorsmall molecule interactions

Biochemical properties of Paracoccus denitrificans FnrP:Reactions with molecular oxygen and nitric oxide

In Paracoccus denitrificans, three CRP/FNR family regulatory proteins, NarR, NnrR and FnrP, control the switch between aerobic and anaerobic (denitrification) respiration. FnrP is a [4Fe-4S] cluster containing homologue of the archetypal O2 sensor FNR from E. coli and accordingly regulates genes encoding aerobic and anaerobic respiratory enzymes in response to O2, and also NO, availability. Here we show that FnrP undergoes O2-driven [4Fe-4S] to [2Fe-2S] cluster conversion that involves up to 2 O2 per cluster, with significant oxidation of released cluster sulfide to sulfane observed at higher O2 concentrations. The rate of the cluster reaction was found to be ~6-fold lower than that of E. coli FNR, suggesting that FnrP can remain transcriptionally active under microaerobic conditions. This is consistent with a role for FnrP in activating expression of the high O2 affinity cytochrome c oxidase under microaerobic conditions. Cluster conversion resulted in dissociation of the transcriptionally active FnrP dimer into monomers. Therefore, along with E. coli FNR, FnrP belongs to the subset of FNR proteins in which cluster type is correlated with association state. Interestingly, two key charged residues, Arg140 and Asp154, that have been shown to play key roles in the monomer-dimer equilibrium in E. coli FNR are not conserved in FnrP, indicating that different protomer interactions are important for this equilibrium. Finally, the FnrP [4Fe-4S] cluster is shown to undergo reaction with multiple NO molecules, resulting in iron nitrosyl species and dissociation into monomers

Sensing mechanisms of iron–sulfur cluster regulatory proteins elucidated using native mass spectrometry

The ability to sense and respond to various key environmental cues is important for the survival and adaptability of many bacteria, including pathogens. The particular sensitivity of iron–sulfur (Fe–S) clusters is exploited in nature, such that multiple sensor-regulator proteins, which coordinate the detection of analytes with a (in many cases) global transcriptional response, are Fe–S cluster proteins. The fragility and sensitivity of these Fe–S clusters make studying such proteins difficult, and gaining insight of what they sense, and how they sense it and transduce the signal to affect transcription, is a major challenge. While mass spectrometry is very widely used in biological research, it is normally employed under denaturing conditions where non-covalently attached cofactors are lost. However, mass spectrometry under conditions where the protein retains its native structure and, thus, cofactors, is now itself a flourishing field, and the application of such ‘native’ mass spectrometry to study metalloproteins is now relatively widespread. Here we describe recent advances in using native MS to study Fe–S cluster proteins. Through its ability to accurately measure mass changes that reflect chemistry occurring at the cluster, this approach has yielded a remarkable richness of information that is not accessible by other, more traditional techniques

Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O-2

The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2

Crystal Structure of the Transcription Regulator RsrR Reveals a [2Fe-2S] Cluster Coordinated by Cys, Glu and His Residues

The recently discovered Rrf2 family transcriptional regulator RsrR coordinates a [2Fe-2S] cluster. Remarkably, binding of the protein to RsrR-regulated promoter DNA sequences is switched on and off through the facile cycling of the [2Fe-2S] cluster be-tween +2 and +1 states. Here, we report high resolution crystal structures of the RsrR dimer, revealing that the [2Fe-2S] cluster is asymmetrically coordinated across the RsrR monomer-monomer interface by two Cys residues from one subunit and His and Glu residues from the other. To our knowledge, this is the first example of a protein bound [Fe-S] cluster with three different amino acid side chains as ligands, and of Glu acting as ligand to a [2Fe-2S] cluster. Analyses of RsrR structures revealed a conformation-al change, centered on Trp9, which results in a significant shift in the DNA-binding helix-turn-helix region

Site-specific encoding of photoactivity and photoreactivity into antibody fragments

Design of biomolecules that perform two or more distinct functions in response to light remains challenging. Here, we have introduced concurrent photoactivity and photoreactivity into an epidermal growth factor receptor (EGFR)-targeting antibody fragment, 7D12. This was achieved by site-specific incorporation of photocaged tyrosine (pcY) for photoactivity and p-benzoyl-ʟ-phenylalanine (Bpa) for photoreactivity into 7D12. We identified a position for installing Bpa in 7D12 that has minimal effect on 7D12–EGFR binding affinity in the absence of light. Upon exposure to 365-nm light, this Bpa-containing 7D12 mutant forms a covalent bond with EGFR in an antigen-specific manner. We then developed a method for site-specific incorporation of pcY and Bpa at two distinct sites in 7D12. Finally, we demonstrated that in the absence of light, this pcY- and Bpa-containing mutant of 7D12 does not bind to EGFR, but irradiation with 365-nm light activates (1) specific binding and (2) covalent bond formation with EGFR