Nitric Oxide Sensing by the [4Fe-4S] Regulator ArnR

What the study examined

ArnR (anaerobic respiration regulator) controls the aerobic/anaerobic respiratory switch in Corynebacterium glutamicum, a Gram-positive actinobacterium in the same suborder (Corynebacterineae) as the pathogens Corynebacterium diphtheriae and Mycobacterium tuberculosis. Earlier genetic work established that ArnR represses the narKGHJI operon (respiratory nitrate reductase) and the hmp gene (an NO-detoxifying flavohemoglobin) under aerobic conditions, and that this repression is relieved when nitric oxide (NO) accumulates. What remained unresolved was the physical cofactor and chemistry behind ArnR's sensing.

A 2025 study in Metallomics by Crack, Harvey, Johnson and Le Brun purified and characterized ArnR biochemically to define its metal cofactor and its reactivity toward oxygen and NO. The authors combined UV-visible and circular dichroism spectroscopy, native electrospray ionization mass spectrometry (native MS), inductively coupled plasma mass spectrometry (ICP-MS), analytical gel filtration, and DNA-binding assays (electrophoretic mobility shift assays and surface plasmon resonance using the ReDCaT approach), supported by AlphaFold3 structural modeling and Foldseek analysis.

Key findings: a [4Fe-4S] cluster that resists O2 but reacts with NO

ArnR is a homodimer that binds one [4Fe-4S] cluster per subunit, with an absorbance signature consistent with such clusters (ε at 420 nm of roughly 13.5 mM-1 cm-1). Cluster ligation involves three conserved cysteines (Cys179, Cys193, Cys223) plus an unidentified fourth ligand. The holo (cluster-bound) form binds promoter DNA tightly, with a dissociation constant of about 30 nM for the ArnR dimer at the hmp promoter.

Crucially, the [4Fe-4S] cluster is unusually resistant to oxygen, degrading only very slowly in air. This oxygen stability explains a longstanding puzzle: ArnR keeps repressing its target genes under aerobic conditions rather than falling apart in the presence of O2. By contrast, the cluster reacts readily with NO. Nitrosylation at low NO stoichiometry (roughly two or fewer NO per cluster) produces mono- and dinitrosylated species. Native MS showed that the mononitrosyl dimer retained partial DNA binding, whereas the dinitrosyl form no longer bound promoter DNA at all.

The mechanism: nitrosylation switches off DNA binding

The results define ArnR as an iron-sulfur nitrosative-stress sensor that works by cluster nitrosylation rather than by direct oxygen chemistry. When NO reacts with the [4Fe-4S] cluster, it forms nitrosyl-iron adducts (of the dinitrosyl-iron-complex / Roussin's-ester family common to iron-sulfur NO sensors). Progressive nitrosylation to the dinitrosyl state changes the protein such that ArnR releases the DNA and repression is lifted.

A notable feature is that DNA release does not require complete destruction of the cluster; even partial nitrosylation is sufficient to modulate binding. Because physiological NO is likely sub-micromolar, the high sensitivity of the mono- to dinitrosyl transition allows graded, titratable derepression. This positions ArnR alongside other [4Fe-4S] and [2Fe-2S] NO-responsive regulators (such as NsrR, WhiD/WhiB-family proteins, and FNR) whose clusters act as sacrificial or reversible NO reaction centers to reprogram transcription.

The physiological logic is indirect sensing of anaerobiosis. ArnR does not measure O2 directly. Instead, low oxygen permits NO -- generated as a byproduct of basal nitrate/nitrite reduction -- to persist in the cytoplasm; that accumulated NO nitrosylates ArnR and derepresses both the nitrate-reductase machinery (narKGHJI) needed for anaerobic respiration and the flavohemoglobin (hmp) needed to clear the very NO that is building up.

How it connects to the metal-microbiome-disease axis

ArnR is a concrete example of how a metal cofactor -- here an iron-sulfur cluster -- is the physical device a microbe uses to read its chemical environment and rewire gene expression. Nitric oxide is not only a bacterial metabolite; it is a central weapon of host innate immunity, produced by inducible nitric oxide synthase (iNOS) in macrophages and in the gut mucosa. Iron-sulfur NO sensors like ArnR, and the related NsrR and WhiB-type regulators in the closely allied Mycobacterium and Corynebacterium pathogens, let bacteria detect host-derived nitrosative stress and switch on defenses such as flavohemoglobin (Hmp), which converts NO to relatively harmless nitrate.

This links directly to iron biology and nutritional immunity. Assembly of the [4Fe-4S] cluster depends on bioavailable iron, so the same host strategies that restrict microbial iron (and the disruptions to metal handling caused by heavy-metal exposure) can influence whether NO-sensing regulators like ArnR are functional at all. In the metal-microbiome-disease framing, metal status governs iron-sulfur metalloregulators, those regulators govern how gut and pathogenic bacteria survive nitrosative and low-oxygen stress, and that survival shapes community composition and host-microbe conflict. ArnR itself was characterized in an industrial/soil organism, so the disease relevance is by homology to its pathogenic relatives and by the shared chemistry of iron-sulfur NO sensing -- a mechanistic bridge, not a claim that ArnR drives human disease directly.