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

Abstract

In Ralstonia eutropha H16, two genes, norA and norB, form a dicistronic operon that is controlled by the NO-responsive transcriptional regulator NorR. NorB has been identified as a membrane-bound NO reductase, but the physiological function of NorA is unknown. We found that, in a NorA deletion mutant, the promoter activity of the norAB operon was increased 3-fold, indicating that NorA attenuates activation of NorR. NorA shows limited sequence similarity to the oxygen carrier hemerythrin, which contains a di-iron center. Indeed, optical and EPR spectroscopy of purified NorA revealed the presence of a di-iron center, which binds oxygen in a similar way as hemerythrin. Nitric oxide (NO)2 is an obligate intermediate of denitrification (1, 2), formed by the reduction of nitrite by nitrite reductases and subsequently converted to nitrous oxide by NO reductases. 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. Orthologs of NorA are present in many genomes of proteobacteria and firmicutes (5) and have been annotated as DnrN (Pseudomonas stutzeri), ScdA (Staphylococcus aureus), or YtfE (Escherichia coli and Salmonella enterica). The expression of DnrN, ScdA, and YtfE has been shown to be up-regulated by NO or nitrosating agents (6 -9). The non-denitrifiers S. aureus, E. coli, and S. enterica, however, do not possess the norB gene, which demonstrates that norA-like genes do not necessarily co-occur with norB. The NO-dependent expression of norA and its coexpression with norB in R. eutropha suggest a function for NorA in NO metabolism. However, it was shown previously that a nonpolar in-frame deletion of the norA gene did not affect denitrification of R. eutropha cells in terms of growth, accumulation of nitrite or nitrous oxide, or formation of dinitrogen (4). In this study we report the purification and characterization of NorA from R. eutropha. We show that NorA is an NO-binding di-iron protein that modulates the NOresponsive expression of the norAB operon in denitrifying cells of R. eutropha. EXPERIMENTAL PROCEDURESStrains and Plasmids-Strains and plasmids used in this study are listed in Table 1. E. coli JM109 was used for propagation of plasmids. E. coli S17-1 served as the donor in conjugative transfers. E. coli BL21(DE3) was used for overproduction of NorAhexahistidine fusion protein. Mutant strains of R. eutropha were constructed by a gene replacement strategy (15) using the suicide vector pLO1, and were verified by Southern blot analysis. The NorR, NorA, and NorB proteins referred to in this study are encoded by the norR1, norA1, and norB1 genes located on megaplasmid pHG1 of R. eutropha. A chromosomally encoded paralog nor gene cluster termed norR2A2B2 has not been extensively studied, but mutational analyses indicate that either of the two nor clusters is sufficient for denitrification (4).All R. eutropha strains used in this stu...

cis- and trans-acting elements involved in regulation of norB (norZ), the gene encoding nitric oxide reductase in Neisseria gonorrhoeae

“…It has also been suggested that YtfE may play an important role in the biogenesis and repair of stress-damaged Fe-S clusters (Justino et al, 2006(Justino et al, , 2007. In R. eutropha, norA is believed to be a cytoplasmic NO-binding protein that regulates gene transcription by lowering the free cytoplasmic concentration of NO (Strube et al, 2007). We confirmed that dnrN is regulated by NsrR, as its aerobic expression increased 11-fold in the DnsrR mutant (Fig.…”

Widespread Distribution in Pathogenic Bacteria of Di-Iron Proteins That Repair Oxidative and Nitrosative Damage to Iron-Sulfur Centers

“…The average values for the two strains were indistinguishable, 162 (Ϯ18) nmol of NO reduced min Ϫ1 ⅐ mg of bacterial dry mass Ϫ1 for the mutant compared with 164 (Ϯ46) nmol of NO reduced min Ϫ1 ⅐ mg of bacterial dry mass Ϫ1 for the parental strain. Furthermore, these rates of NO reduction were sufficiently high to exclude the possibility that NO accumulates to a higher concentration in cultures of the mutant, causing more severe or even different types of damage (50).…”

“…An NO-sensitive electrode was used to eliminate an alternative possibility, i.e., that the dnrN mutant is defective in its ability to reduce NO compared with the parent strain (50). The rates of NO reduction by bacteria harvested from these cultures were measured.…”

Internal Sense of Direction: Sensing and Signaling from Cytoplasmic Chemoreceptors

“…PBPb domains are high-affinity small-molecule binding domains characterized in ABC transporters (57). Hemerythrins bind and respond to oxygen through coordinated iron atoms (80,81) and have also been reported to mediate responses to nitric oxide (82). NMT1 domains have been characterized for their role in the synthesis of the pyrimidine moiety of thiamine and are regulated by thiamine (83,84).…”