Differentiated, Promoter-specific Response of [4Fe-4S] NsrR DNA Binding to Reaction with Nitric Oxide

Abstract

NsrR is an iron-sulfur cluster protein that regulates the nitric oxide (NO) stress response of many bacteria. NsrR from Streptomyces coelicolor regulates its own expression and that of only two other genes, hmpA1 and hmpA2, which encode HmpA enzymes predicted to detoxify NO. NsrR binds promoter DNA with high affinity only when coordinating a [4Fe-4S] cluster. Here we show that reaction of [4Fe-4S] NsrR with NO affects DNA binding differently depending on the gene promoter. Binding to the hmpA2 promoter was abolished at ∼2 NO per cluster, although for the hmpA1 and nsrR promoters, ∼4 and ∼8 NO molecules, respectively, were required to abolish DNA binding. Spectroscopic and kinetic studies of the NO reaction revealed a rapid, multi-phase, non-concerted process involving up to 8–10 NO molecules per cluster, leading to the formation of several iron-nitrosyl species. A distinct intermediate was observed at ∼2 NO per cluster, along with two further intermediates at ∼4 and ∼6 NO. The NsrR nitrosylation reaction was not significantly affected by DNA binding. These results show that NsrR regulates different promoters in response to different concentrations of NO. Spectroscopic evidence indicates that this is achieved by different NO-FeS complexes.

13. Reactivity of iron-sulfur clusters with nitric oxide

“…Figures 9 and 10 show representative data for NsrR (before and after addition of NO) from some of these techniques (see section 2), illustrating their application. Resonance Raman data were consistent with an Fe-S cluster coordinated by three cysteines and one oxygen-containing residue, rather than a histidine residue as found in IscR [58,68]. Moreover, some low molecular weight thiols (such as DTT) were found to modify the [4Fe-4S] cluster [58,126], drastically reducing its O2 stability leading to rapid disassembly into a [2Fe-2S] [58].…”

“…The binding interaction between NsrR and the hmpA1 promoter was the tightest, with full binding observed at a ratio of [4Fe-4S] NsrR monomer to DNA of approximately 2:1 (i.e. one NsrR dimer per DNA) [68]. This is significantly tighter than that previously reported for the [2Fe-2S] form, for which full binding of hmpA1 promoter was not observed even with a several hundred-fold excess of protein [58].…”

“…~420 nm) and to the newly formed iron-nitrosyl (e.g. ~360 nm) [41,67,68]. This has revealed that the nitrosylation reaction of Fe-S cluster regulatory proteins is a multi-step reaction, involving multiple (8-10) NO molecules per cluster, which reaches completion within a few seconds.…”

“…While CymR and SaiR represent regulators that do not contain a co-factor [122,123], IscR, RirA, RsrR and NsrR contain three conserved cysteine residues (Figure 7) that have been shown, or are predicted, to bind an Fe-S cluster, with the type, [2Fe-2S] or [4Fe-4S], being dependent on the particular regulator [58,68,74,124,125]. For example, IscR and RsrR are known to contain a [2Fe-2S] cluster, whereas NsrR from both Bacillus subtilis and Streptomyces coelicolor has been shown to bind a [4Fe-4S] cluster [58,68,74,125,126]. In all examples, where data are available, the Fe-S functions as the sensory module.…”

“…Nitrosylation was also investigated optically, by measuring changes in the absorbance and circular dichroism (CD) properties of the Fe-S cluster [68]. Following the sequential addition of sub-stoichiometric amounts of NO, changes in the absorption bands of the cluster were observed.…”