Binding of a single nitric oxide molecule is sufficient to disrupt DNA binding of the nitrosative stress regulator NsrR.

Abstract

The regulatory protein NsrR, a member of the Rrf2 protein superfamily, plays a major role in the cellular response to nitrosative stress in many benign and pathogenic bacteria. The homodimeric protein binds a [4Fe-4S] cluster in each subunit (termed holo NsrR), and represses transcription of genes primarily involved in NO detoxification. Holo NsrR reacts rapidly with multiple NO molecules per [4Fe-4S] cluster, via a complex reaction, with loss of DNA binding and formation of NsrR-bound iron-nitrosyl species. However, the point at which DNA binding is lost is unknown. Here, we demonstrate using surface plasmon resonance (SPR) and native mass spectrometry (MS) that holo NsrR binds the promoter regions of NsrR-regulated genes with promoter-dependent nanomolar affinity, while hemi-apo NsrR (i.e. one cluster per dimer) binds >10-fold less tightly, and the cluster-free (apo) form not at all. Strikingly, native MS provided detailed information about the reaction of NO with the physiologically relevant form of NsrR, i.e. DNA-bound dimeric NsrR. Reaction with a single NO molecule per NsrR dimer is sufficient to abolish DNA binding. This exquisite sensitivity of DNA binding to NO is consistent with the importance of de-repressing NO detoxification systems at the earliest opportunity to minimise damage due to nitrosative stress. Furthermore, the data show that previously characterised iron-nitrosyls, which form at higher ratios of NO to [4Fe-4S], are not physiologically relevant for regulating the NsrR on/off switch.