Nitric oxide (NO) is a ubiquitous biomolecule that is known as a signaling compound in eukaryotes and prokaryotes. In addition, NO is involved in all conversions of the biogeochemical nitrogen cycle: denitrification, nitrification, and the anaerobic oxidation of ammonium (Anammox). Until now, NO has not been measured with high spatial resolution within microbial communities, such as biofilms, sediments, aggregates, or microbial mats, because the available sensors are not robust enough and their spatial resolution is insufficient. Here we describe the fabrication and application of a novel Clark-type NO microsensor with an internal reference electrode and a guard anode. The NO microsensor has a spatial resolution of 60-80 μm, a sensitivity of 2 pA μM-1, and a detection limit of ∼30 nM. Hydrogen sulfide (H2S) was found to be a major interfering compound for the electrochemical detection of NO. The application of the novel NO microsensor to nitrifying biofilms and marine sediments revealed dynamic NO concentration profiles with peaks in the oxic parts of the samples. The local concentrations suggested that NO may be an important bioactive compound in natural environments. The consumption and production of NO occurs in separate regions of stratified microbial communities and indicates that it is linked to distinct biogeochemical cycles. © 2008 American Chemical Society.

Only three biological pathways are known to produce oxygen: photosynthesis, chlorate respiration and the detoxification of reactive oxygen species. Here we present evidence for a fourth pathway, possibly of considerable geochemical and evolutionary importance. The pathway was discovered after metagenomic sequencing of an enrichment culture that couples anaerobic oxidation of methane with the reduction of nitrite to dinitrogen. The complete genome of the dominant bacterium, named ĝ€̃ Candidatus Methylomirabilis oxyferaĝ€™, was assembled. This apparently anaerobic, denitrifying bacterium encoded, transcribed and expressed the well-established aerobic pathway for methane oxidation, whereas it lacked known genes for dinitrogen production. Subsequent isotopic labelling indicated that ĝ€̃ M. oxyferaĝ€™ bypassed the denitrification intermediate nitrous oxide by the conversion of two nitric oxide molecules to dinitrogen and oxygen, which was used to oxidize methane. These results extend our understanding of hydrocarbon degradation under anoxic conditions and explain the biochemical mechanism of a poorly understood freshwater methane sink. Because nitrogen oxides were already present on early Earth, our finding opens up the possibility that oxygen was available to microbial metabolism before the evolution of oxygenic photosynthesis. © 2010 Macmillan Publishers Limited. All rights reserved.

In catalyzing the reversible hydration of CO2 to bicarbonate and protons, the ubiquitous enzyme carbonic anhydrase (CA) plays a crucial role in CO2 transport, in acid-base balance, and in linking local acidosis to O2 unloading from hemoglobin. Considering the structural similarity between bicarbonate and nitrite, we hypothesized that CA uses nitrite as a substrate to produce the potent vasodilator nitric oxide (NO) to increase local blood flow to metabolically active tissues. Here we show that CA readily reacts with nitrite to generate NO, particularly at low pH, and that the NO produced in the reaction induces vasodilation in aortic rings. This reaction occurs under normoxic and hypoxic conditions and in various tissues at physiological levels of CA and nitrite. Furthermore, two specific inhibitors of the CO2 hydration, dorzolamide and acetazolamide, increase the CA-catalyzed production of vasoactive NO from nitrite. This enhancing effect may explain the known vasodilating effects of these drugs and indicates that CO2 and nitrite bind differently to the enzyme active site. Kinetic analyses show a higher reaction rate at high pH, suggesting that anionic nitrite participates more effectively in catalysis. Taken together, our results reveal a novel nitrous anhydrase enzymatic activity of CA that would function to link the in vivo main end products of energy metabolism (CO2/H+) to the generation of vasoactive NO. The CA-mediated NO production may be important to the correlation between blood flow and metabolic activity in tissues, as occurring for instance in active areas of the brain. Copyright © 2009 the American Physiological Society.

Denitrification supports anoxic growth of Pseudomonas aeruginosa in infections. Moreover, denitrification may provide oxygen (O2) resulting from dismutation of the denitrification intermediate nitric oxide (NO) as seen in Methylomirabilis oxyfera. To examine the prevalence of NO dismutation we studied O2 release by P. aeruginosa in airtight vials. P. aeruginosa rapidly depleted O2 but NO supplementation generated peaks of O2 at the onset of anoxia, and we demonstrate a direct role of NO in the O2 release. However, we were not able to detect genetic evidence for putative NO dismutases. The supply of endogenous O2 at the onset of anoxia could play an adaptive role when P. aeruginosa enters anaerobiosis. Furthermore, O2 generation by NO dismutation may be more widespread than indicated by the reports on the distribution of homologues genes. In general, NO dismutation may allow removal of nitrate by denitrification without release of the very potent greenhouse gas, nitrous oxide.