NsrR from Streptomyces coelicolor is a Nitric Oxide-Sensing [4Fe-4S] Cluster Protein with a Specialized Regulatory Function [Enzymology]

March 14th, 2015 by Crack, J. C., Munnoch, J., Dodd, E. L., Knowles, F., Al Bassam, M. M., Kamali, S., Holland, A. A., Cramer, S. P., Hamilton, C. J., Johnson, M. K., Thomson, A. J., Hutchings, M. I., Le Brun, N. E.

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, Mossbauer, resonance Raman spectroscopies and native mass spectrometry demonstrate that S. 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 flavohaemoglobins, indicating that ScNsrR has a specialized regulatory function focussed on NO-detoxification, and is not a global regulator like some NsrR orthologues. EMSAs and DNaseI 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 E85 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.