Nitric Oxide Sensing by [4Fe–4S] ArnR: NO Disrupts DNA Binding

Overview

This paper dissects how the Corynebacterium glutamicum transcriptional regulator ArnR uses an iron–sulfur cofactor to sense nitric oxide (NO) generated during nitrate respiration and thereby modulates expression of nitrate reductase (narKGHJI) and flavohemoglobin (hmp), with implications for nitrosative stress management relevant to host-associated Corynebacteria.

What was studied and how?

This original mechanistic study examined purified C. glutamicum ArnR as the primary matrix (protein in buffered solution; ArnR–DNA complexes with the hmp promoter). The microbial system is dimeric ArnR that represses narKGHJI and hmp; the central metal species is a protein-bound [4Fe–4S]^2+ cluster (Fe oxidation state formally mixed-valent), with minor Zn detected. A suite of analytical methods quantified cluster identity, stoichiometry, and reactivity: UV–visible spectroscopy and circular dichroism (CD) to track Fe–S/iron–nitrosyl optical signatures; native electrospray ionization mass spectrometry (native ESI-MS) and LC-MS for cofactor/oligomer states and ArnR–DNA complexes; inductively coupled plasma–MS for elemental content; surface plasmon resonance (SPR) and electrophoretic mobility shift assays (EMSA) to define promoter binding and affinity. Oxygen reactivity was probed by exposing 22 µM [4Fe–4S] to ~220 µM dissolved O2; NO reactivity used proline/DEA NONOate titrations across 0–≥8 NO per cluster. An AlphaFold3 model and gel filtration established dimerization and domain architecture, and a working regulatory model was proposed.

Most important findings

ArnR is a constitutive dimer that natively binds one [4Fe–4S]+ per subunit and specifically recognizes hmp promoter DNA with K_d = 30 ± 3 nM (as dimer; 60 nM per cluster) by SPR. As-isolated ArnR contained 3.8–4.2 Fe and 4.1 S per monomer and ~0.4–0.6 Zn (ε_420 = 13.5 ± 0.5 mM^−1 cm^−1). The [4Fe–4S] cluster is O2-resistant on the hour timescale at ~220 µM O2, consistent with aerobic repression in vivo. NO induces rapid, stepwise nitrosylation: ~1–2 NO per cluster generates a chiral intermediate and weakens DNA binding; di-nitrosylated [4Fe–4S] dimers fail to bind DNA, without requiring full cluster degradation. Iron-nitrosyl end-products akin to RRE/RBS arise only at ≥8 NO per cluster and are not necessary for regulatory switching.

Strengths

Methodological triangulation is a major strength: orthogonal optical (UV–vis, CD), compositional (ICP-MS), structural/stoichiometric (native ESI-MS), and biophysical (SPR, EMSA) measurements coherently define the [4Fe–4S] species, oligomeric state, and promoter binding. Quantitative affinity (nanomolar K_d) was obtained under reducing, anaerobic conditions appropriate for Fe–S proteins. Native MS directly visualized ArnR–DNA complexes and their NO-dependent dissociation while resolving mono- versus di-nitrosylated states, strengthening causal inference about speciation and function. O2 and NO challenges were executed with explicit analyte stoichiometry, enabling speciation–function mapping.

Any Limitations

All experiments were performed with recombinant ArnR and synthetic promoter fragments in vitro; direct intracellular concentrations of NO during nitrate respiration were not measured here. The fourth ligand to the [4Fe–4S] cluster remains unidentified, and the physiological significance of the minor Zn signal is unresolved. EMSA data were qualitative due to probe size, placing emphasis on SPR/native-MS for quantitation.

Future Perspectives

Clinically relevant Corynebacteria encounter fluctuating O2/NO in host tissues; this work positions ArnR as an NO-responsive gatekeeper of nitrate respiration and NO detoxification. Immediate next steps grounded in the data include resolving the cluster ligation environment (to pinpoint the non-Cys ligand), determining kinetics and reversibility of 4Fe–4S formation on promoter-bound ArnR, and mapping ArnR’s in vivo promoter occupancy across O2/NO regimes. Establishing whether di-nitrosylation is reversible under cytosolic conditions, and quantifying ArnR control of hmp/narKGHJI expression dynamics, could inform strategies to modulate nitrosative stress tolerance in industrial strains or opportunistic pathogens. Integration with cAMP-dependent GlxR signaling suggests potential for combinatorial control of respiration and stress pathways.

Citation

Crack JC, Harvey LR, Johnson KE, Le Brun NE. ArnR binds a [4Fe–4S] cluster and indirectly senses anaerobicity in Corynebacteria. Metallomics. 2025;17:mfaf026. doi:10.1093/mtomcs/mfaf026