Nitric oxide (NO) is a ubiquitous signalling molecule and cytotoxic agent. In order to survive and thrive, organisms must be able to respond to NO, and the regulatory protein NsrR, a member of the Rrf2 protein superfamily, plays a major role in this as it is found in a wide range of benign and pathogenic bacteria. NsrR binds a [4Fe-4S] cluster as a cofactor and, in the absence of NO, represses transcription of genes primarily involved NO detoxification. Previously, we have shown that [4Fe-4S] NsrR reacts rapidly with NO, in a complex reaction involving multiple NO molecules per cluster, resulting in formation of NsrR-bound iron-nitrosyl species [3]. Here, we sought to investigate the reaction of [4Fe-4S] NsrR bound to DNA, in order to determine when high affinity DNA-binding is lost. Using surface plasmon resonance (SPR) and native mass spectrometry (MS) NsrR binding to the promoter regions of two NsrR-regulated genes, hmpA1 and hmpA2 was probed, revealing the nature of binding and the reaction of DNA-bound NsrR with NO in remarkable detail.