Background Carbonic anhydrases (CAs) are universal metalloenzymes that catalyze the reversible conversion of carbon dioxide (CO2) and bicarbonate (HCO3-). They are involved in various biological processes, including pH control, respiration, and photosynthesis. To date, eight evolutionarily unrelated classes of CA families (α, β, γ, δ, ζ, η, θ, and ι) have been identified. All are characterized by an active site accommodating the binding of a metal cofactor, which is assumed to play a central role in catalysis. This feature is thought to be the result of convergent evolution. Results Here, we report that a previously uncharacterized protein group, named “COG4337,” constitutes metal-independent CAs from the newly discovered ι-class. Genes coding for COG4337 proteins are found in various bacteria and photosynthetic eukaryotic algae. Biochemical assays demonstrated that recombinant COG4337 proteins from a cyanobacterium (Anabaena sp. PCC7120) and a chlorarachniophyte alga (Bigelowiella natans) accelerated CO2 hydration. Unexpectedly, these proteins exhibited their activity under metal-free conditions. Based on X-ray crystallography and point mutation analysis, we identified a metal-free active site within the cone-shaped α+β barrel structure. Furthermore, subcellular localization experiments revealed that COG4337 proteins are targeted into plastids and mitochondria of B. natans, implicating their involvement in CO2 metabolism in these organelles. Conclusions COG4337 proteins shared a short sequence motif and overall structure with ι-class CAs, whereas they were characterized by metal independence, unlike any known CAs. Therefore, COG4337 proteins could be treated as a variant type of ι-class CAs. Our findings suggested that this novel type of ι-CAs can function even in metal-poor environments (e.g., the open ocean) without competition with other metalloproteins for trace metals. Considering the widespread prevalence of ι-CAs across microalgae, this class of CAs may play a role in the global carbon cycle.
A continuous-flow method for measuring atmospheric HNO2 concentration in real time has been developed that uses a chemiluminescent NOx monitor. A Na2CO3 solution strips gaseous HNO2 from the atmosphere by means of pulling an air sample and the solution through a glass coil and mixing continuously with ascorbic acid solution which reduces nitrite to NO. The mixture is led into a gas-liquid separating coil consisting of microporous PTFE tubing. The NO evolved from the separating coil is swept out by a stream of clean air and detected with a chemiluminescent NOx monitor. The technique utilizes a dual flow system and dual channel NOx monitor to correct positive interferences from NO2 and peroxyacetyinitrate (PAN). The concentration of HNO2 is determined by difference between the two measurements. Sensitivity of the method is a function of the ratio of sampling flow rate to carrier gas flow rate, which permits readily a highly sensitive measurement.
The determination of nitrite and nitrate in aqueous environmental samples is of considerable interest in research concerning hydrological chemistry, chemical oceanography, and atmospheric chemistry. Nitrate is an essential nutrient which controls the biomass in natural water; the amount also indicates the extent of pollution and eutrophication of the aquatic environment. Nitrite is a nitrogen-cycle intermediate and a useful indicator of the equilibrium state of the oxidative and reductive pathways of the nitrogen cycle. Also, nitric acid and nitrous acid are very important trace constituents of the atmosphere in both the gaseous and particulate phases. Nitric acid is a major NOx sink in photochemical air pollution. Nitrous acid is recognized as a major source of a very reactive hydroxyl radical, which is a key intermediate for all photochemical oxidant formations. These gases and particulates are precursors of acid rain, and are deposited on the ground mainly by wet precipitation, such as rain and snow.Among many analytical methods proposed for the determination of nitrate and nitrite, a widely used method for analyses of natural water samples is based on the reduction of nitrate to nitrite and a subsequent spectrophotometric determination via the Griess reaction, 1-3 which involves the diazotization of sulfanilic acid and successive coupling with 1-naphthylamine. Though numerous modifications have been suggested, primarily based on changing either the reagent being diazotized or coupled, sulfanilamide or sulfanilic acid and N-(1-naphtyl)ethylenediamine are the most popular couples. 4,5 The spectrophotometric method can provide reliable results for natural water samples, but suffers from interferences from suspended and colored substances in the samples. Cox 6 proposed a chemiluminescence analysis technique, which is more highly sensitive and free from these interferences. The technique is based on the chemical reduction of nitrate and nitrite to NO and detection via a chemiluminescence reaction of NO with ozone. Garside 7 first applied this technique to seawater analyses. Braman and Hendrix 8 reported on an application used for environmental water samples. These methods used NaI for reducing nitrite to NO, and iron(II)-molybdate or vanadium(III) for reducing nitrate to NO. The reduction procedures in these methods require highly acidic conditions and/or operation at a relatively high temperature. In the monitoring method of ambient gaseous HNO2, 9 we employed ascorbic acid in a dilute H2SO4 for reducing nitrite to NO. This reagent is easy to handle and the reaction is rapid and quantitative at low nitrite levels. By reducing nitrate to nitrite with an alkaline hydrazine solution, the measurement system was further developed to a gaseous HNO3 monitoring method. 10The coupling of the chemiluminescence detection with a flowinjection technique (FIA) would permit the development of sensitive, precise, rapid, and high throughput methodologies having a wide dynamic range. Aoki and Wakabayashi 11 reported a chemilumine...
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