Title
Differentiated, promoter-specific response of [4Fe-4S] NsrR DNA-binding to reaction with nitric oxide
Author
Jason C. Crack, Dimitri A. Svistunenko, John Munnoch, Andrew J. Thomson, Matthew I. Hutchings, Nick E. Le Brun
Year
2016
Journal
The Journal of Biological Chemistry
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 thehmpA2 promoter was abolished at ∼2 NO per cluster, although for the hmpA1 and nsrRpromoters, ∼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.
Full Article
Instrument
J-810
Keywords
Circular dichroism, Ligand binding, Biochemistry, Inorganic chemistry