Redox‐Controlled Dinitrosyl Formation at the Diiron‐Oxo Center of NorA

Cramm, Rainer; Strube, Katja

doi:10.1016/s0076-6879(07)37006-7

Cited by 5 publications

(14 citation statements)

References 29 publications

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“… 27 , 28 We note that the R. eutropha YtfE orthologue NorA has been reported to reduce nitrite to NO in vitro. ( 38 ) Thus, YtfE may function as a NO-generating nitrite reductase. In vivo experiments were conducted to test this possibility .…”

Section: Resultsmentioning

confidence: 99%

“…This purification strategy also leads to the formation of intramolecular (between Cys30 and Cys31) and intermolecular disulfide bonds, the latter resulting in YtfE dimers. 23 , 32 , 38 Therefore, we chose to purify YtfE under anaerobic conditions.…”

Section: Resultsmentioning

confidence: 99%

“…In the presence of ascorbate (+60 mV versus standard hydrogen electrode (SHE) at pH 7.0) and phenazine methosulfate (+80 mV), 48 the R. eutropha YtfE orthologue NorA was able to catalyze a limited number of nitrite reductions. 38 , 39 The reaction was slow; the K m (∼7 mM) for nitrite was high and was subject to inhibition by NO. In contrast, E. coli YtfE has been reported to reduce NO to N 2 O in the presence of ascorbate and N , N , N ′, N ′-tetramethyl- p -phenylenediamine (+276 mV), or NADH (−320 mV), albeit rather slowly.…”

Section: Resultsmentioning

confidence: 99%

“…Accordingly, the R. eutropha YtfE orthologue NorA has been reported to attenuate the ability of NorR transcriptional regulator to detect NO in vivo. ( 38 , 39 , 66 , 67 ) In contrast, a YtfE-dependent release of [4Fe-4S] NsrR transcriptional repression was recently demonstrated in E. coli . ( 27 ) Therefore, we investigated the in vitro effect of nitrite and NO on the [4Fe-4S] cluster of Streptomyces coelicolor NsrR (ScNsrR) in the presence of YtfE.…”

Section: Resultsmentioning

confidence: 99%

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The Di-Iron Protein YtfE Is a Nitric Oxide-Generating Nitrite Reductase Involved in the Management of Nitrosative Stress

et al. 2022

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Previously characterized nitrite reductases fall into three classes: siroheme-containing enzymes (NirBD), cytochrome c hemoproteins (NrfA and NirS), and copper-containing enzymes (NirK). We show here that the di-iron protein YtfE represents a physiologically relevant new class of nitrite reductases. Several functions have been previously proposed for YtfE, including donating iron for the repair of iron–sulfur clusters that have been damaged by nitrosative stress, releasing nitric oxide (NO) from nitrosylated iron, and reducing NO to nitrous oxide (N 2 O). Here, in vivo reporter assays confirmed that Escherichia coli YtfE increased cytoplasmic NO production from nitrite. Spectroscopic and mass spectrometric investigations revealed that the di-iron site of YtfE exists in a mixture of forms, including nitrosylated and nitrite-bound, when isolated from nitrite-supplemented, but not nitrate-supplemented, cultures. Addition of nitrite to di-ferrous YtfE resulted in nitrosylated YtfE and the release of NO. Kinetics of nitrite reduction were dependent on the nature of the reductant; the lowest K m , measured for the di-ferrous form, was ∼90 μM, well within the intracellular nitrite concentration range. The vicinal di-cysteine motif, located in the N-terminal domain of YtfE, was shown to function in the delivery of electrons to the di-iron center. Notably, YtfE exhibited very low NO reductase activity and was only able to act as an iron donor for reconstitution of apo-ferredoxin under conditions that damaged its di-iron center. Thus, YtfE is a high-affinity, low-capacity nitrite reductase that we propose functions to relieve nitrosative stress by acting in combination with the co-regulated NO-consuming enzymes Hmp and Hcp.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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The Di-Iron Protein YtfE Is a Nitric Oxide-Generating Nitrite Reductase Involved in the Management of Nitrosative Stress

et al. 2022

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“…A second protective mechanism involves NO-inducible Repair of Iron Centers (RIC) di-iron proteins. RICs use a hemerythrin-like domain to decrease oxidative damage by interacting with iron storage proteins like Dps and IscU to deliver iron to damaged Fe-S clusters (36,(40)(41)(42)(43)(44).…”

Section: Introductionmentioning

confidence: 99%

NorA, HmpX, and NorB cooperate to reduce NO toxicity during denitrification and plant pathogenesis in Ralstonia solanacearum

Truchon

Hendrich

Bigott

et al. 2021

Preprint

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Ralstonia solanacearum, which causes bacterial wilt disease of many crops, needs denitrifying respiration to succeed inside its plant host. In the hypoxic environment of plant xylem vessels this pathogen confronts toxic oxidative radicals like nitric oxide (NO), which is generated by both bacterial denitrification and host defenses. R. solanacearum has multiple distinct mechanisms that could mitigate this stress, including Repair of Iron Cluster (RIC) homolog NorA, nitric oxide reductase NorB, and flavohaemoglobin HmpX. During denitrification and tomato pathogenesis and in response to exogenous NO, R. solanacearum upregulated norA, norB, and hmpX. Single mutants lacking ΔnorB, ΔnorA, or ΔhmpX increased expression of many iron and sulfur metabolism genes, suggesting that losing even one NO detoxification system demands metabolic compensation. Single mutants suffered only moderate fitness reductions in host plants, possibly because they upregulated their remaining detoxification genes. However, ΔnorA/norB, ΔnorB/hmpX, and ΔnorA/hmpX double mutants grew poorly in denitrifying culture and in planta. Loss of norA, norB, and hmpX may be lethal, since the methods used to construct the double mutants did not generate a triple mutant. Aconitase activity assays showed that NorA, HmpX and especially NorB are important for maintaining iron-sulfur cluster proteins. Additionally, plant defense genes were upregulated in tomatoes infected with the NO-overproducing ΔnorB mutant, suggesting that bacterial detoxification of NO reduces pathogen visibility. Thus, R. solanacearum’s three NO detoxification systems each contribute to and are collectively essential for overcoming metabolic oxidative stress during denitrification, for virulence and growth in tomato, and for evading host plant defenses.ImportanceThe soilborne plant pathogen Ralstonia solanacearum (Rs) causes bacterial wilt, a serious and widespread threat to global food security. Rs is metabolically adapted to low oxygen conditions, using denitrifying respiration to survive in the host and cause disease. However, bacterial denitrification and host defenses generate nitric oxide (NO), which is toxic and also alters signaling pathways in both plants and the pathogen. Rs mitigates NO with a trio of mechanistically distinct proteins: NO-reductase NorB, Repair of Iron Centers NorA, and oxidoreductase HmpX. This redundancy, together with analysis of mutants and in-planta dual transcriptomes, indicates that maintaining low NO levels is integral to Rs fitness in tomatoes (because NO damages iron-cluster proteins) and to evading host recognition (because bacterially produced NO can trigger plant defenses).

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