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The molecular structure of the flavohemoglobin from Alcaligenes eutrophus has been determined to a resolution of 1.75 A and refined to an R‐factor of 19.6%. The protein comprises two fused modules: a heme binding module, which belongs to the globin family, and an FAD binding oxidoreductase module, which adopts a fold like ferredoxin reductase. The most striking deviation of the bacterial globin structure from those of other species is the movement of helix E in a way to provide more space in the vicinity of the distal heme binding site. A comparison with other members of the ferredoxin reductase family shows similar tertiary structures for the individual FAD and NAD binding domains but largely different interdomain orientations. The heme and FAD molecules approach each other to a minimal distance of 6.3 A and adopt an interplanar angle of 80 degrees. The electron transfer from FAD to heme occurs in a predominantly polar environment and may occur directly or be mediated by a water molecule.

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Ralstonia eutropha is a strictly respiratory facultative lithoautotrophic β-proteobacterium. In the absence of organic substrates, H₂ and CO₂ are used as sole sources of energy and carbon. In the absence of oxygen, the organism can respire by denitrification. The recent determination of the complete genome sequence of strain H16 provides the opportunity to reconcile the results of previous physiological and biochemical studies in light of the coding capacity. These analyses revealed genes for several isoenzymes, permit assignment of well-known physiological functions to previously unidentified genes, and suggest the presence of unknown components of energy metabolism. The respiratory chain is fueled by two NADH dehydrogenases, two uptake hydrogenases and at least three formate dehydrogenases. The presence of genes for five quinol oxidases and three cytochrome oxidases indicates that the aerobic respiration chain adapts to varying concentrations of dioxygen. Several additional components may act in balancing or dissipation of redox energy. Paralogous sets of nitrate reductase and nitric oxide reductase genes result in enzymatic redundancy for denitrification.

SummaryRalstonia eutropha H16 mediates the reduction of nitric oxide (NO) to nitrous oxide (N 2 O) with two isofunctional single component membrane-bound NO reductases (NorB1 and NorB2). This reaction is integrated into the denitrification pathway that involves the successive reduction of nitrate to dinitrogen. The norB1 gene is co-transcribed with norA1 from a s 54 (RpoN)-dependent promoter, located upstream of norA1. With the aid of norA1 H ± lacZ transcriptional fusions and the generation of regulatory mutants, it was shown that norB1 gene transcription requires a functional rpoN gene and the regulator NorR, a novel member of the NtrC family of response regulators. The regulator gene maps adjacent to norAB, is divergently transcribed and present in two copies on the megaplasmid pHG1 (norR1) and the chromosome (norR2). Transcription activation by NorR responds to the availability of NO. A nitrite reductase-deficient mutant that is incapable of producing NO endogenously, showed a 70% decrease of norA1 expression. Addition of the NO-donating agent sodium nitroprusside caused induction of norA1 H ± lacZ transcription. Truncation of the N-terminal receiver domain of NorR1 interrupted the NO signal transduction and led to a constitutive expression of norA1 H ±lacZ. The results indicate that NorR controls the reductive conversion of NO in R. eutropha. This reaction is not strictly co-ordinated on the regulatory level with the other nitrogen oxide-reducing steps of the denitrification chain that are independent of NorR.

Two genes, norB and norZ, encoding two independent nitric oxide reductases have been identified in Alcaligenes eutrophus H16. norB and norZ predict polypeptides of 84.5 kDa with amino acid sequence identity of 90%. While norB resides on the megaplasmid pHG1, the norZ gene is located on a chromosomal DNA fragment. Amino acid sequence analysis suggests that norB and norZ encode integral membrane proteins composed of 14 membrane-spanning helices. The region encompassing helices 3 to 14 shows similarity to the NorB subunit of common bacterial nitric oxide reductases, including the positions of six strictly conserved histidine residues. Unlike the Nor enzymes characterized so far from denitrifying bacteria, NorB and NorZ of A. eutrophus contain an amino-terminal extension which may form two additional helices connected by a hydrophilic loop of 203 amino acids. The presence of a NorB/NorZ-like protein was predicted from the genome sequence of the cyanobacterium Synechocystis sp. strain PCC6803. While the common NorB of denitrifying bacteria is associated with a second cytochrome c subunit, encoded by the neighboring gene norC, the nor loci of A. eutrophus and Synechocystis lack adjacent norC homologs. The physiological roles of norB and norZ in A. eutrophus were investigated with mutants disrupted in the two genes. Mutants bearing single-site deletions in norB or norZ were affected neither in aerobic nor in anaerobic growth with nitrate or nitrite as the terminal electron acceptor. Inactivation of both norB and norZ was lethal to the cells under anaerobic growth conditions. Anaerobic growth was restored in the double mutant by introducing either norB or norZ on a broad-host-range plasmid. These results show that the norB and norZ gene products are isofunctional and instrumental in denitrification.

Nitric oxide (NO) reductase was purified from Ralstonia eutropha (formerly Alcaligenes eutrophus) using a two step chromatographic procedure. Unlike the common NO reductases, the enzyme consists of a single subunit of 75 kDa which contains both high-spin and low-spin heme b, but lacks heme c. One additional iron atom, probably a ferric non-heme iron, was identified per enzyme molecule. Whereas reduced cytochrome c was ineffective as electron donor, NO was reduced at a specific activity of 2.3 W Wmol/min per mg of protein in the presence of 2-methyl-1,4-naphthoquinol.z 1999 Federation of European Biochemical Societies.

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...