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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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Rrf2 family transcription factor NsrR controls expression of genes in a wide range of bacteria in response to nitric oxide (NO). The precise form of the NO-sensing module of NsrR is the subject of controversy because NsrR proteins containing either [2Fe-2S] or [4Fe-4S] clusters have been observed previously. Optical, Mössbauer, resonance Raman spectroscopies and native mass spectrometry demonstrate that Streptomyces coelicolor NsrR (ScNsrR), previously reported to contain a [2Fe-2S] cluster, can be isolated containing a [4Fe-4S] cluster. ChIP-seq experiments indicated that the ScNsrR regulon is small, consisting of only hmpA1, hmpA2, and nsrR itself. The hmpA genes encode NO-detoxifying flavohemoglobins, indicating that ScNsrR has a specialized regulatory function focused on NO detoxification and is not a global regulator like some NsrR orthologues. EMSAs and DNase I footprinting showed that the [4Fe-4S] form of ScNsrR binds specifically and tightly to an 11-bp inverted repeat sequence in the promoter regions of the identified target genes and that DNA binding is abolished following reaction with NO. Resonance Raman data were consistent with cluster coordination by three Cys residues and one oxygen-containing residue, and analysis of ScNsrR variants suggested that highly conserved Glu-85 may be the fourth ligand. Finally, we demonstrate that some low molecular weight thiols, but importantly not physiologically relevant thiols, such as cysteine and an analogue of mycothiol, bind weakly to the [4Fe-4S] cluster, and exposure of this bound form to O2 results in cluster conversion to the [2Fe-2S] form, which does not bind to DNA. These data help to account for the observation of [2Fe-2S] forms of NsrR., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

WhiD is required for the late stages of sporulation in the Gram-positive bacterium Streptomyces coelicolor. WhiD is a member of the WhiB-like family of putative transcription factors that are present throughout the actinomycetes but absent from other organisms. This family of proteins has four near-invariant cysteines, suggesting that these residues might act as ligands for a metal cofactor. Overexpressed WhiD, purified from Escherichia coli, contained substoichiometric amounts of iron and had an absorption spectrum characteristic of a [2Fe-2S] cluster. After Fe-S cluster reconstitution under anaerobic conditions, WhiD contained ∼4 iron atoms/monomer and similar amounts of sulfide ion and gave an absorption spectrum characteristic of a [4Fe-4S] cluster. Reconstituted WhiD gave no electron paramagnetic resonance signal as prepared but, after reduction with dithionite, gave an electron paramagnetic resonance signal (g ∼2.06, 1.94) consistent with a one-electron reduction of a [4Fe-4S]2+ cluster to a [4Fe-4S]1+ state with electron spin of S = ½. The anaerobically reconstituted [4Fe-4S] cluster was oxygen sensitive. Upon exposure to air, absorption at 410 and 505 nm first increased and then showed a steady decrease with time until the protein was colorless in the near UV/visible region. These changes are consistent with an oxygen-induced change from a [4Fe-4S] to a [2Fe-2S] cluster, followed by complete loss of cluster from the protein. Each of the four conserved cysteine residues, Cys-23, -53, -56, and -62, was essential for WhiD function in vivo. [ABSTRACT FROM AUTHOR]