Characterization of the Signaling Domain of the NO-Responsive Regulator NorR from Ralstonia eutropha H16 by Site-Directed Mutagenesis

Abstract

In Ralstonia eutropha H16, the nitric oxide (NO)-responsive transcriptional activator NorR controls the expression of a dicistronic operon that encodes a membrane-bound NO reductase, NorB, and a protein of unknown function, NorA. The N-terminal domain (NTD) of NorR is responsible for perception of the signal molecule, nitric oxide. Thirteen out of 29 conserved residues of the NTD were exchanged by site-directed mutagenesis. Replacement of R63, R72, D93, D96, C112, D130, or F137 strongly decreased NorR-dependent promoter activation, while the exchange of Y95 or H110 led to an increase in promoter activity compared to that of the wild type. A purified truncated NorR comprising only the NTD (NorR-NTD) contained one iron atom per molecule and was able to bind NO in the as-isolated state. Based on the iron content of NorR-NTD proteins with single amino acid replacements, residues R72, D93, D96, C112, and D130 are likely candidates for iron ligands. Residues R63, Y95, and H110 appear not to be involved in NO binding but may take part in subsequent steps of the signal transduction mechanism of NorR.The diatomic radical nitric oxide (NO) is an obligate intermediate of bacterial denitrification. NO is produced from nitrite by respiratory nitrite reductase and is converted to nitrous oxide by nitric oxide reductase (30). Also, nondenitrifiers like Escherichia coli K-12 can produce considerable amounts of NO during growth under hypoxic conditions with nitrate (6). Since NO and derived reactive nitrogen oxides act as toxic and mutagenic agents, defense mechanisms must be induced under conditions of nitrosative stress. NO-responsive transcription factors are found in the Fnr/Crp family (e.g., DnrD [31]), the Rrf2 family (e.g., NsrR [2]), and the NtrC/NifA family (e.g., NorR). The last protein has been identified in the betaproteobacterium Ralstonia eutropha H16 (21) and E. coli (15). However, genomic analysis revealed that NorR orthologs are widely distributed among beta-and gammaproteobacteria (22). Like all proteins of the 54 -dependent NtrC/NifA superfamily (25), NorR shows a typical modular structure, including an N-terminal domain (NTD), a central AAAϩ domain, and a helix-turn-helix domain for DNA binding (3,21). In a recent study, it was shown that the NTD of E. coli NorR harbors a mono-iron center that is able to bind NO as a ferrous nitrosyl (10). Those authors presented a model for the activation of NorR which postulates the existence of an inactive form of NorR, where the ATPase activity of the AAAϩ domain is blocked by the NTD. Upon binding of NO, this block is relieved and NorR switches to an active state which is competent to induce promoter activation. This hypothesis is corroborated by the fact that truncated forms of NorR from E. coli and R. eutropha that lack the NTD show signal-independent activation of gene expression (10, 21). To date, neither the iron ligands nor the residues that are involved in the switching mechanism have been identified.Given the high positional identity (45%) between the amino acid ...

Analysis of the Nitric Oxide-sensing Non-heme Iron Center in the NorR Regulatory Protein

“…Potentially, the near axial species exhibited by the Y98L substitution in NorR, which is similar to the wild-type and Y98F spectra, might represent the activated "ON" state of NorR. In contrast to our results with E. coli NorR, the corresponding Y95L mutation in R. eutropha NorR retains the ability to activate transcription (58,59). Although the contribution of the tyrosine to the {Fe(NO)} 7 unit could be context-dependent, differences in the organism and culture conditions might lead to an increase in the axial species in vivo for Y95L in R. eutropha compared with Y98L in E. coli.…”

“…As mentioned above, these substitutions also have a different phenotype to that of D99A in that transcriptional activation by NorR is constitutive. In contrast, Asp to Asn substitutions in equivalent positions in R. eutropha NorR inactivate the protein (59). At the early stages of structural modeling, it was realized that the side chain of Arg-75 could be constrained to reveal a covalent bond between one of its side-chain nitrogen atoms and the iron atom.…”

Nitrous oxide production and consumption: regulation of gene expression by gas-sensitive transcription factors

“…Formation of a mono-nitrosyl complex at this centre disrupts an intra-molecular interaction, by which the GAF domain inhibits the activity of the AAA þ domain in the absence of NO [53,54]. The nonhaem iron is believed to be coordinated by the side chains of three aspartate residues, an arginine and a cysteine [55,56].…”

Formation of a Dinitrosyl Iron Complex by NorA, a Nitric Oxide-binding Di-iron Protein from Ralstonia eutropha H16

“…In the denitrifier Ralstonia eutropha H16, formation of the NO reductase NorB is controlled by NorR, an NObinding transcriptional activator (3,4). The norB gene is cotranscribed with norA that encodes a protein of unknown function.…”

“…Physiological Characterization-To determine if the presence of NorA alters the level of cytoplasmic NO in denitrifying R. eutropha, an NO-sensitive reporter system was constructed by using the transcriptional activator NorR. Because NorR responds to NO (3,4), the availability of free NO in the cytoplasm of denitrifying R. eutropha cells is reflected by the activity of the NorR-dependent promoter of the norAB operon, PnorA. A single copy of a PnorA-lacZ fusion was established in both the wild-type and the NorA mutant, and promoter activation by NorR was recorded as ␤-galactosidase activity expressed in Miller units.…”