A two-faced molecule offers NO explanation: the proximal binding of nitric oxide to haem

Cytochrome c´ (cyt c´) is found in the periplasmic space of denitrifying bacteria where it is thought to mediate the transfer of NO between the nitrogen-cycle enzymes dissimilatory nitrite reductase and nitric oxide reductase. It contains a 5-coordinate (5c) His-ligated haem that shares spectroscopic and ligand-binding properties with the haem group in the sensory domain of soluble guanylate cyclase (sGC). The latter is an extremely important enzyme involved in the control of vasodilation and blood clotting. Curiously, the enzyme is activated up to 200-fold by the binding of NO to the haem, whereas the binding of CO gives rise to only a mild stimulation of activity. Through X-ray crystallography we have studied NO and CO binding to cyt c´. CO binds to the distal face to give a 6-coordinate (6c) adduct. By contrast, NO binding gives rise to a 5c adduct through the displacement of the proximal His, to give a novel and unexpected proximal binding mode for NO. These results are also supported by a range of spectroscopies. In the absence of a crystal structure for sGC we propose that cyt c´ provides a structural model for the haem domain of this enzyme and thereby helps to explain the differential effects of NO and CO on its activity.

Purification, Characterization, and Genetic Analysis of Cu-Containing Dissimilatory Nitrite Reductase from a Denitrifying Halophilic Archaeon, Haloarcula marismortui

ABSTRACT Cu-containing dissimilatory nitrite reductase (CuNiR) was purified from denitrifying cells of a halophilic archaeon, Haloarcula marismortui. The purified CuNiR appeared blue in the oxidized state, possessing absorption peaks at 600 and 465 nm in the visible region. Electron paramagnetic resonance spectroscopy suggested the presence of type 1 Cu (gII = 2.232; AII = 4.4 mT) and type 2 Cu centers (gII = 2.304; AII = 13.3 mT) in the enzyme. The enzyme contained two subunits, whose apparent molecular masses were 46 and 42 kDa, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis indicated that the two subunits were identical, except that the 46-kDa subunit was 16 amino acid residues longer than the 42-kDa subunit in the N-terminal region. A nirK gene encoding the CuNiR was cloned and sequenced, and the deduced amino acid sequence with a residual length of 361 amino acids was homologous (30 to 41%) with bacterial counterparts. Cu-liganding residues His-133, Cys-174, His-182, and Met-187 (for type 1 Cu) and His-138, His-173, and His-332 (for type 2 Cu) were conserved in the enzyme. As generally observed in the halobacterial enzymes, the enzymatic activity of the purified CuNiR was enhanced during increasing salt concentration and reached its maximum in the presence of 2 M NaCl with the value of 960 μM NO2 − · min−1 · mg−1.

Dissimilatory Nitrite Reductase Genes from Autotrophic Ammonia-Oxidizing Bacteria

ABSTRACT The presence of a copper-containing dissimilatory nitrite reductase gene (nirK) was discovered in several isolates of β-subdivision ammonia-oxidizing bacteria using PCR and DNA sequencing. PCR primers Cunir3 and Cunir4 were designed based on published nirK sequences from denitrifying bacteria and used to amplify a 540-bp fragment of the nirK gene fromNitrosomonas marina and five additional isolates of ammonia-oxidizing bacteria. Amplification products of the expected size were cloned and sequenced. Alignment of the nucleic acid and deduced amino acid (AA) sequences shows significant similarity (62 to 75% DNA, 58 to 76% AA) between nitrite reductases present in these nitrifiers and the copper-containing nitrite reductase found in classic heterotrophic denitrifiers. While the presence of a nitrite reductase in Nitrosomonas europaea is known from early biochemical work, preliminary sequence data from its genome indicate a rather low similarity to the denitrifier nirKs. Phylogenetic analysis of the partial nitrifier nirK sequences indicates that the topology of the nirK tree corresponds to the 16S rRNA andamoA trees. While the role of nitrite reduction in the metabolism of nitrifying bacteria is still uncertain, these data show that the nirK gene is present in closely related nitrifying isolates from many oceanographic regions and suggest thatnirK sequences retrieved from the environment may include sequences from ammonia-oxidizing bacteria.

Denitrification within the genus Azospirillum and other associative bacteria

This paper originates from an address at the 8th International Symposium on Nitrogen Fixation with Non-Legumes, Sydney, NSW, December 2000 Different Azospirillumstrains and some other plant growth-promoting rhizobacteria (PGPR) were screened for the occurrence of genes coding for denitrification and nitrogenase reductase (nifH) using polymerase chain reaction (PCR)-based techniques. All PGPR examined were nitrogenase-positive. Azospirillum strains were remarkably dissimilar with respect to denitrification capabilities, in particular with respect to genes of the dissimilatory nitrite reductase. A. brasilense, A. lipoferum and A. halopraeferens strains possess a cytochrome cd1-containing nitrite reductase with low sequence similarities among them. A. irakense and A. doebereinerae have a Cu-containing nitrite reductase and A. amazonense is unable to denitrify. The molecular data were corroborated by activity measurements. The current results indicate that the inability to perform denitrification is unlikely a selective advantage for Azospirillum spp. and other associative bacteria for forming an association with plant roots.

Denitrification by Actinomycetes and Purification of Dissimilatory Nitrite Reductase and Azurin fromStreptomyces thioluteus

ABSTRACT Many actinomycete strains are able to convert nitrate or nitrite to nitrous oxide (N2O). As a representative of actinomycete denitrification systems, the system of Streptomyces thioluteus was investigated in detail. S. thioluteusattained distinct cell growth upon anaerobic incubation with nitrate or nitrite with concomitant and stoichiometric conversion of nitrate or nitrite to N2O, suggesting that the denitrification acts as anaerobic respiration. Furthermore, a copper-containing, dissimilatory nitrite reductase (CuNir) and its physiological electron donor, azurin, were isolated. This is the first report to show that denitrification generally occurs among actinomycetes.