Nitrous oxide (N2O) is estimated to contribute about 6% of the global warming effect due to greenhouse gases. N2O is also predicted to be the single most important ozone-depleting emission in the twenty-first century. Great progress has been made in N2O measurement, but there is a critical need for sensors that can be used to map the spatial variation of N2O emissions over a wide area. In this article, we outline where N2O measurement is required, describe advances that have been made in developing sensitive analytical techniques and review some promising new technologies. Our aim is to assist both those new to N2O measurement, enabling them to select the most appropriate of the available technology, and to inform those developing new analytical techniques

The ability to persist in the absence of growth triggered by low-oxygen levels is a critical process for the survival of mycobacterial species in many environmental niches. MSMEG_5243 (fsq), a gene of unknown function in Mycobacterium smegmatis, is up-regulated in response to hypoxia and regulated by DosRDosS/DosT, an oxygen- and redox-sensing two-component system that is highly conserved in mycobacteria. In this communication, we demonstrate that MSMEG_5243 is a flavin-sequestering protein and henceforth refer to it as Fsq. Using an array of biochemical and structural analyses, we show that Fsq is a member of the diverse superfamily of flavin- and deazaflavin-dependent oxidoreductases (FDORs) and is widely distributed in mycobacterial species. We created a markerless deletion mutant of fsq and demonstrate that fsqis required for cell survival during hypoxia. Using fsq deletion and overexpression, we found that fsq enhances cellular resistance to hydrogen peroxide treatment. The X-ray crystal structure of Fsq, solved to 2.7 Å, revealed a homodimeric organization with FAD bound noncovalently. The Fsq structure also uncovered no potential substrate-binding cavities, as the FAD is fully enclosed, and electrochemical studies indicated that the Fsq:FAD complex is relatively inert and does not share common properties with electron-transfer proteins. Taken together, our results suggest that Fsq reduces the formation of reactive oxygen species (ROS) by sequestering free FAD during recovery from hypoxia, thereby protecting the cofactor from undergoing autoxidation to produce ROS. This finding represents a new paradigm in mycobacterial adaptation to hypoxia.

Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased K-m(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (similar to 60 and 200 s(-1), respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.