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

Serrano, Pauline N.; Wang, Hongxin; Crack, Jason C.; Prior, Christopher; Hutchings, Matthew I.; Thomson, Andrew J.; Kamali, Saeed; Yoda, Yoshitaka; Zhao, Jiyong; Hu, Michael Y.; Ercan, E.; Oganesyan, Vasily S.; Brun, Nick E. Le; Cramer, Stephen P.

doi:10.1002/anie.201607033

Cited by 34 publications

(40 citation statements)

References 46 publications

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“…23 It therefore appears the protein-bound Roussin’s red ester, once overlooked due to its EPR-silent nature in the neutral form, is indeed more common than previously thought. 22–26 …”

Section: Resultsmentioning

confidence: 99%

“…18–20 However, there is growing evidence that other iron nitrosyl species, such as the Roussin’s red ester, [( μ -Cys) 2 Fe 2 (NO) 4 ], a structural analogue of the red iron nitrosyl salt reported by Roussin in 1858 (Figure 1), 21 are more common than previously thought. 22–26 While recent studies have focused on the nitrosylation of transcription factors, the [Fe 4 S 4 ] clusters of DNA repair enzymes, such as endonuclease III (EndoIII) and DinG, are also targets for NO reactivity. 27,28 …”

Section: Introductionmentioning

confidence: 99%

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Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe₄S₄] Cluster Nitrosylation

et al. 2018

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Here we characterize the [Fe4S4] cluster nitrosylation of a DNA repair enzyme, endonuclease III (EndoIII), using DNA-modified gold electrochemistry and protein film voltammetry, electrophoretic mobility shift assays, mass spectrometry of whole and trypsin-digested protein, and a variety of spectroscopies. Exposure of EndoIII to nitric oxide under anaerobic conditions transforms the [Fe4S4] cluster into a dinitrosyl iron complex, [(Cys)2 Fe(NO)2]-, and Roussin’s red ester, [(μ-Cys)2Fe2(NO)4], in a 1:1 ratio with an average retention of 3.05 ± 0.01 Fe per nitrosylated cluster. The formation of the dinitrosyl iron complex is consistent with previous reports, but the Roussin’s red ester is an unreported product of EndoIII nitrosylation. Hyperfine sublevel correlation (HYSCORE) pulse EPR spectroscopy detects two distinct classes of NO with 14N hyperfine couplings consistent with the dinitrosyl iron complex and reduced Roussin’s red ester. Whole-protein mass spectrometry of EndoIII nitrosylated with 14NO and 15NO support the assignment of a protein-bound [(μ-Cys)2Fe2(NO)4] Roussin’s red ester. The [Fe4S4]2+/3+ redox couple of DNA-bound EndoIII is observable using DNA-modified gold electrochemistry, but nitrosylated EndoIII does not display observable redox activity using DNA electrochemistry on gold despite having a similar DNA-binding affinity as the native protein. However, direct electrochemistry of protein films on graphite reveals the reduction potential of native and nitrosylated EndoIII to be 127 ± 6 and −674 ± 8 mV vs NHE, respectively, corresponding to a shift of approximately −800 mV with cluster nitrosylation. Collectively, these data demonstrate that DNA-bound redox activity, and by extension DNA-mediated charge transport, is modulated by [Fe4S4] cluster nitrosylation.

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“…23 It therefore appears the protein-bound Roussin’s red ester, once overlooked due to its EPR-silent nature in the neutral form, is indeed more common than previously thought. 22–26 …”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe₄S₄] Cluster Nitrosylation

et al. 2018

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“…An exception to this was the recent detection of NO binding to the human mitochondrial protein Miner2, but in this case the NO complex was stable and no further reaction was observed. Progress in identifying the products of nitrosylation was made recently through the application of NRVS, which revealed both RRE and RBS‐like species for both NsrR and WhiD, with significant amounts of DNIC species also present in NsrR . Furthermore, LC‐MS studies of NsrR recently demonstrated the precise nature of the RRE‐species formed .…”

Section: Discussionmentioning

confidence: 99%

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

Crack

Brun

2019

Chemistry A European J

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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.

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“…Speculation about a possible role of nitric oxide as a regulatory ligand for WhiB and other Wbl proteins was stimulated by the extremely high affinity for nitric oxide of some Wbl proteins of streptomycetes and mycobacteria, which has been studied in detail at the molecular level 80– 83 . It has generally been implied that the source of nitric oxide would be exogenous (e.g.…”

Section: New Perspectives In Growth and Development Of Streptomycesmentioning

confidence: 99%

Recent advances in understanding Streptomyces

2016

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About 2,500 papers dated 2014–2016 were recovered by searching the PubMed database for Streptomyces, which are the richest known source of antibiotics. This review integrates around 100 of these papers in sections dealing with evolution, ecology, pathogenicity, growth and development, stress responses and secondary metabolism, gene expression, and technical advances. Genomic approaches have greatly accelerated progress. For example, it has been definitively shown that interspecies recombination of conserved genes has occurred during evolution, in addition to exchanges of some of the tens of thousands of non-conserved accessory genes. The closeness of the association of Streptomyces with plants, fungi, and insects has become clear and is reflected in the importance of regulators of cellulose and chitin utilisation in overall Streptomyces biology. Interestingly, endogenous cellulose-like glycans are also proving important in hyphal growth and in the clumping that affects industrial fermentations. Nucleotide secondary messengers, including cyclic di-GMP, have been shown to provide key input into developmental processes such as germination and reproductive growth, while late morphological changes during sporulation involve control by phosphorylation. The discovery that nitric oxide is produced endogenously puts a new face on speculative models in which regulatory Wbl proteins (peculiar to actinobacteria) respond to nitric oxide produced in stressful physiological transitions. Some dramatic insights have come from a new model system for Streptomyces developmental biology, Streptomyces venezuelae, including molecular evidence of very close interplay in each of two pairs of regulatory proteins. An extra dimension has been added to the many complexities of the regulation of secondary metabolism by findings of regulatory crosstalk within and between pathways, and even between species, mediated by end products. Among many outcomes from the application of chromosome immunoprecipitation sequencing (ChIP-seq) analysis and other methods based on “next-generation sequencing” has been the finding that 21% of Streptomyces mRNA species lack leader sequences and conventional ribosome binding sites. Further technical advances now emerging should lead to continued acceleration of knowledge, and more effective exploitation, of these astonishing and critically important organisms.

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

Cited by 34 publications

References 46 publications

Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe₄S₄] Cluster Nitrosylation

Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe₄S₄] Cluster Nitrosylation

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

Recent advances in understanding Streptomyces

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

Cited by 34 publications

References 46 publications

Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe4S4] Cluster Nitrosylation

Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe4S4] Cluster Nitrosylation

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

Recent advances in understanding Streptomyces

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Product

Resources

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Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe₄S₄] Cluster Nitrosylation

Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe₄S₄] Cluster Nitrosylation

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