Biological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme composed of two separately purifiable component proteins encoded by the structural genes nifH, nifD, and nifK. Deletion of the Azotobacter vinelandii nifS gene lowers the activities of both nitrogenase component proteins. Because both nitrogenase component proteins have metallocluster prosthetic groups that are composed of iron-and sulfur-containing cores, this result indicated that the nifS gene product could be involved in the mobilization of the iron or sulfur required for metallocluster formation. In the present work, it is shown that NIFS is a pyridoxal phosphatecontaining homodimer that catalyzes the formation ofL-alanine and elemental sulfur by using L-cysteine as substrate. NIFS activity is extremely sensitive to thiol-specific alkylating reagents, which indicates the participation of a cysteinyl thiolate at the active site. Based on these results we propose that an enzyme-bound cysteinyl persulfide that requires the release of the sulfur from the substrate L-cysteine for its formation ultimately provides the inorganic sulfide required for nitrogenase metaflocluster formation. The recent discovery of nifSlike genes in non-nitrogen-fiing organisms also raises the possibility that the reaction catalyzed by NIIFS represents a universal mechanism that involves pyridoxal phosphate chemistry, in the mobilization of the sulfur required for metailocluster formation.The reduction in both A. vinelandii nitrogenase component protein activities as a result of nijS deletion, which has also been reported for Klebsiella pneumoniae (6), could not be attributed to a regulatory effect (3). Thus, because both Fe protein and MoFe protein activities were affected by deletion of nijf , and the common feature of both component proteins is that they contain metalloclusters, we have considered that NIFS might be involved in the acquisition or mobilization of the inorganic Fe or sulfur required for metallocluster formation. This possibility is supported by the observation that cell pellets of diazotrophically grown A. vinelandii nifS mutants are pale tan rather than the characteristic dark brown of wild type. The dark color of diazotrophically grown wild-type A. vinelandii is attributed to the metalloclusters contained within the nitrogenase component proteins that form a substantial portion of the soluble protein fraction.As a strategy to elucidate the function of the NIFS polypeptide, we chose to produce large amounts of it in Escherichia coli to facilitate its purification and biochemical characterization. In the present study, we have purified the NIFS protein, demonstrated that a specific reaction is catalyzed by NIFS, and proposed a biochemical function for NIFS activity in relation to metallocluster formation. In addition, we discuss the possibility that the reaction catalyzed by NIFS represents a universal mechanism for the mobilization of the sulfur required for metallocluster formation. Many proteins that have important electron tr...

The nifU gene product is required for the full activation of the metalloenzyme nitrogenase, the catalytic component of biological nitrogen fixation. In the present work, a hybrid plasmid that contains the Azotobacter vinelandii nifU gene was constructed and used to hyperexpress the NIFU protein in Escherichia coli. Recombinant NIFU was purified to homogeneity and was found to be a homodimer of 33-kDa subunits with approximately two Fe atoms per subunit. The combination of UV/visible absorption, variable-temperature magnetic circular dichroism, EPR, and resonance Raman spectroscopies shows the presence of a [2Fe-2S]2+,+ center (Em = -254 mV) with complete cysteinyl coordination in each subunit. The electronic, magnetic, and vibrational properties of the [2Fe-2S]2+,+ center do not conform to those established for any of the spectroscopically distinct types of 2Fe ferredoxins. These distinctive properties appear to be a consequence of a novel arrangement of coordinating cysteinyl residues in NIFU, and the residues likely to be involved in cluster coordination are discussed in light of primary sequence comparisons to other putative [2Fe-2S] proteins. The observed physicochemical properties of NIFU and its constituent [2Fe-2S] cluster also provide insight into the role of this protein in nitrogenase metallocluster biosynthesis.

The NifS and NifU nitrogen fixation-specific gene products are required for the full activation of both the Fe-protein and MoFe-protein of nitrogenase from Azotobacter vinelandii. Because the two nitrogenase component proteins both require the assembly of [Fe-S]-containing clusters for their activation, it has been suggested that NifS and NifU could have complementary functions in the mobilization of sulfur and iron necessary for nitrogenase-specific [Fe-S] cluster assembly. The NifS protein has been shown to have cysteine desulfurase activity and can be used to supply sulfide for the in vitro catalytic formation of [Fe-S] clusters. The NifU protein was previously purified and shown to be a homodimer with a [2Fe-2S] cluster in each subunit. In the present work, primary sequence comparisons, amino acid substitution experiments, and optical and resonance Raman spectroscopic characterization of recombinantly produced NifU and NifU fragments are used to show that NifU has a modular structure. One module is contained in approximately the N-terminal third of NifU and is shown to provide a labile rubredoxin-like ferric-binding site. Cysteine residues Cys35, Cys62, and Cys106 are necessary for binding iron in the rubredoxin-like mode and visible extinction coefficients indicate that up to one ferric ion can be bound per NifU monomer. The second module is contained in approximately the C-terminal half of NifU and provides the [2Fe-2S] cluster-binding site. Cysteine residues Cys137, Cys139, Cys172, and Cys175 provide ligands to the [2Fe-2S] cluster. The cysteines involved in ligating the mononuclear Fe in the rubredoxin-like site and those that provide the [2Fe-2S] cluster ligands are all required for the full physiological function of NifU. The only two other cysteines contained within NifU, Cys272 and Cys275, are not necessary for iron binding at either site, nor are they required for the full physiological function of NifU. The results provide the basis for a model where iron bound in labile rubredoxin-like sites within NifU is used for [Fe-S] cluster formation. The [2Fe-2S] clusters contained within NifU are proposed to have a redox function involving the release of Fe from bacterioferritin and/or the release of Fe or an [Fe-S] cluster precursor from the rubredoxin-like binding site.

Abstract--The corrosion rates of carbon steel by monocultures and various combinations of aerobic (Bacillus sp.), fermentative (Hafnia alvei) and sulfate-reducing (Desulfovibrio gigas) bacterial biofilms in an aerobic, continuously flowing freshwater reactor containing 0.4 mM sulfate showed marked differences. Biofilm formation and electrode colonization resulted in decreases in the open circuit potential (OCP). The corrosion rate was measured non-destructively as the admittance (1/Rot) by electrochemical impedance spectroscopy (EIS) on a four sided working electrode which allowed estimates of the reproducibility. The monocultures each induced greater corrosion initially but with time the rates of corrosion decreased to that of the sterile control. The D. gigas was unable to grow in the bulk phase but formed an apparently non-living biofilm. The admittance (the measure of corrosion) was greatest when the sulfate-reducing bacterium, D. gigas, was present in a consortium. The consortia containing D. gigas + H. alvei showed a significantly higher corrosion rate than the triculture or the other dicultures.The microbial biomass and the bacterial community structure actually on the electrode surfaces were examined by viable counts, most probable number (MPN) estimations and phospholipid fatty acid (PLFA) profiles determined after extraction and gas chromatographic analysis. The rate of corrosion was not directly related to the total microbial biomass or the number of species on the coupon. The rate of corrosion did not del~end on the ratio of heterotrophic to sulfate-reducing bacteria (SRB) or absolute number of SRB. The P][FA analysis showed the organisms on the working electrodes were more stressed/ starved than when grown in the bulk phase for inocula. This study demonstrates that different combinations of bacteria growing on the same substrata (electrode) in the same bulk phase induce very different corrosion rates. The maximum corrosion rate with the isolate combinations was within 70% of an enrichment with a community composition (determined by PLFA analysis) like that of the native corrosion tubercules.

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From the health stand-point, atmospheric particulate matter (PM) is regulated through PM 10 and PM 2.5 conventions by the Directive 2008/50/EC. The Directive points out the negative impact on human health due to PM 2.5 and recognizes that no threshold has been identified for such pollutant at which no risk is foreseen for the population. Then, the goal is to pursue a general reduction of PM 2.5 . Traditionally, the analytical techniques used to monitor the PM water-soluble inorganic ionic fraction involve filter-based procedures to collect, process, and analyze samples. Data obtained, while accurate, lack temporal resolution. Time resolution is required on the time-scale of the evolution of the planetary boundary layer to understand the processes that govern transport and transformation of atmospheric aerosol. In this paper, we investigated PM 2.5 nitrite, nitrate, sulfate, chloride, sodium, ammonium, calcium, and magnesium ions using a URG 9000-D aerosol ion monitor with 1-h time-resolution and detection limit of 0.1 µg/m 3 . The gas phase is separated from the aerosol phase with a liquid diffusion parallel-plate denuder. Daily trends of the pollutants measured in downtown Rome are discussed and interpreted with reference to atmospheric conditions.

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