Sequence analysis of an internal 9.72-kb segment from the 30-kb denitrification gene cluster of Pseudomonas stutzeri

AbstractThe DNA segment was sequenced that links the nir-nor and nos gene clusters for denitrification of Pseudomonas stutzeri ATCC 14405. Of 10 predicted gene products, four are putative membrane proteins. Sequence similarity was detected with the subunit III of cytochrome-c oxidase (ORF175), PQQ3 of the biosynthetic pathway for pyrrolo-quinoline quinone (ORF393), S-adenosylmethionine-dependent uroporphyrinogen-III C-methyltransferase (ORF278), the cytochrome cd1 nitrite reductase and the NirF protein involved in the biosynthesis of heme d1 (ORF507), LysR type transcriptional regulators (ORF286), short-chain alcohol dehydrogenases (ORF247), and a hypothetical protein, YBEC, of Escherichia coli (ORF57). The current data together with previous work establish a contiguous DNA sequence of 29.2 kb comprising the supercluster of nos-nir-nor genes for denitrification in this bacterium

The nirSTBM region coding for cytochrome cd1-dependent nitrite respiration of Pseudomonas stutzeri consists of a cluster of mono-, di-, and tetraheme proteins

AbstractGenes for respiratory nitrite reduction (denitrification) of Pseudomonas stutzeri are clustered within 7 kbp. A 4.6-kbp Hind III-Kpn I fragment carrying nirS, the structural gene for cytochrome cd1, was sequenced. An open reading frame immediately downstream of nirScodes for a 22.8-kDa protein with four heme c-binding motifs. Mutagenesis of this gene causes an apparent defect in electron donation to cytochrome cd1. Following this ORF are the structural genes for cytochrome c552, cytochrome c551, and ORF5 that codes for a 11.9-kDa monoheme protein. All cytochromes have a signal sequence for protein export

\u3csup\u3e1\u3c/sup\u3eH NMR Studies on the CuA Center of Nitrous Oxide Reductase from \u3cem\u3ePseudomonas stutzeri\u3c/em\u3e

1H NMR spectra of the CuA center of N2OR from Pseudomonas stutzeri, and a mutant enzyme that contains only CuA, were recorded in both H2O- and D2O-buffered solution at pH 7.5. Several sharp, well-resolved hyperfine-shifted 1H NMR signals were observed in the 60 to −10 ppm chemical shift range. Comparison of the native and mutant N2OR spectra recorded in H2O-buffered solutions indicated that several additional signals are present in the native protein spectrum. These signals are attributed to a dinuclear copper(II) center. At least two of the observed hyperfine-shifted signals associated with the dinuclear center, those at 23.0 and 13.2 ppm, are lost upon replacement of H2O buffer with D2O buffer. These data indicate that at least two histidine residues are ligands of a dinuclear Cu(II) center. Comparison of the mutant N2OR 1H NMR spectra recorded in H2O and D2O indicates that three signals, c (27.5 ppm), e (23.6 ppm), and i (12.4 ppm), are solvent exchangeable. The two most strongly downfield-shifted signals (c and e) are assigned to the two Nε2H (N-H) protons of the coordinated histidine residues, while the remaining exchangeable signal is assigned to a backbone N-H proton in close proximity to the CuA cluster. Signal e was found to decrease in intensity as the temperature was increased, indicating that proton e resides on a more solvent-exposed histidine residue. One-dimensional nOe studies at pH 7.5 allowed the histidine ring protons to be definitively assigned, while the remaining signals were assigned by comparison to previously reported spectra from CuA centers. The temperature dependence of the observed hyperfine-shifted 1H NMR signals of mutant N2OR were recorded over the temperature range of 276−315 K. Both Curie and anti-Curie temperature dependencies are observed for sets of hyperfine-shifted protons. Signals a and h (cysteine protons) follow anti-Curie behavior (contact shift increases with increasing temperatures), while signals b−g, i, and j (histidine protons) follow Curie behavior (contact shift decreases with increasing temperatures). Fits of the temperature dependence of the observed hyperfine-shifted signals provided the energy separation (ΔEL) between the ground (2B3u) and excited (2B2u) states. The temperature data obtained for all of the observed hyperfine-shifted histidine ligand protons provided a ΔEL value of 62 ± 35 cm-1. The temperature dependence of the observed cysteine CβH and CαH protons (a and h) were fit in a separate experiment providing a ΔEL value of 585 ± 125 cm-1. The differences between the ΔEL values determined by 1H NMR spectroscopy and those determined by EPR or MCD likely arise from coupling between relatively low-frequency vibrational states and the ground and excited electronic states

Electron paramagnetic resonance studies on nitrogenase. III. Function of magnesium adenosine 5'-triphosphate and adenosine 5'-diphosphate in catalysis by nitrogenase

The electron paramagnetic resonance spectra of azoferredoxin and molybdoferredoxin, components of the nitrogenase of Clostridium pasteurianum, disappear when the proteins are oxidized by certain dyes. When molybdoferredoxin and azoferredoxin were mixed in a 1 to 2 molar ratio, the electron paramagnetic resonance spectrum of the mixture was the sum of the two spectra with the exception of a slight change in the azoferredoxin signal. Addition of magnesium ATP and dithionite to this reconstituted nitrogenase resulted in a rapid change in the spectrum of both nitrogenase components; the molybdoferredoxin spectrum at all g-values decreased with a half-life less than 70 ms to 40% of its original size whereas the azoferredoxin signal changed in shape and size with a half-life of less than 40 ms. If an ATP-generating system was added instead of MgATP so that no ADP accumulated, then the molybdoferredoxin signal almost completely disappeared and the azoferredoxin signal changed in shape and slightly in size. These changes occurred at molar ratios of molybdoferredoxin to azoferredoxin from 1:14 to 1:0.2. If the reaction was allowed to consume the reductant, then the molybdoferredoxin signal(s) was restored but the azoferredoxin signal disappeared. The signal of azoferredoxin was restored and the signal of molybdoferredoxin again disappeared on addition of more reductant. The data suggest that for nitrogenase to catalyze the reduction of substrates, the magnesium ATP-reduced azoferredoxin complex is formed first and this complex then reacts with molybdoferredoxin to allow electron flow. In addition the data suggests that the rate-limiting reaction is an ATP-mediated electron flow from azoferredoxin to molybdoferredoxin. Finally the results show that no flow of electrons from azoferredoxin or molybdoferredoxin occurs when a mixture of ADP and ATP in a molar ratio of 2:1 is added initially or is reached by conversion of ATP to ADP and inorganic phosphate during reduction of protons. A mechanism consistent with these findings is proposed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33941/1/0000208.pd

Electron paramagnetic resonance studies on nitrogenase. II. Interaction of adenosine 5'-triphosphate with azoferredoxin

The interaction of ATP with both iron-sulfur proteins of nitrogenase from Clostridium pasteurianum, azoferredoxin and molybdoferredoxin, has been studied by low-temperature EPR spectroscopy. ATP in the presence of Mg2+ changes the rhombic EPR signal of azoferredoxin with g-values of 2.06, 1.94 and 1.87 to an axial signal, with g values of 2.04 and 1.93. The binding of two molecules of ATP per azoferredoxin dimer (mol. wt 55 000) is suggested. Comparative data with other purine and pyrimidine nucleotides and ATP analogues demonstrate the involvement of structural elements of the substrate in the conversion of the EPR signal of azoferredoxin. A similar effect is induced by 5 M urea, which suggests that ATP causes a conformation change of the protein. In contrast, no effect of ATP was observed on the EPR signal of molybdoferredoxin.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33940/1/0000207.pd

On the structure and function of nitrogenase from W5

Molybdoferredoxin from W5 was fractionated into MoFd with two atoms of molybdenum per 220,000 daltons and a specific activity of 2.6 [mu]moles C2H2 reduced/min/mg protein and into a catalytically inactive species with an identical protein moiety but an incomplete active centre. Native MoFd is a tetramer composed of two 50,000 and two 60,000 dalton subunits. At low protein concentrations the tetramer is in equilibrium with a dimer. Under low ionic strength and at low pH further dissociation into monomers occurs. MoFd and azoferredoxin have distinct electron paramagnetic resonance spectra. The EPR spectrum of AzoFd and that of the combination of the two nitrogenase components undergoes characteristic changes upon addition of MgATP2-.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34037/1/0000314.pd

Perspectives and Integration in SOLAS Science

Why a chapter on Perspectives and Integration in SOLAS Science in this book? SOLAS science by its nature deals with interactions that occur: across a wide spectrum of time and space scales, involve gases and particles, between the ocean and the atmosphere, across many disciplines including chemistry, biology, optics, physics, mathematics, computing, socio-economics and consequently interactions between many different scientists and across scientific generations. This chapter provides a guide through the remarkable diversity of cross-cutting approaches and tools in the gigantic puzzle of the SOLAS realm. Here we overview the existing prime components of atmospheric and oceanic observing systems, with the acquisition of ocean–atmosphere observables either from in situ or from satellites, the rich hierarchy of models to test our knowledge of Earth System functioning, and the tremendous efforts accomplished over the last decade within the COST Action 735 and SOLAS Integration project frameworks to understand, as best we can, the current physical and biogeochemical state of the atmosphere and ocean commons. A few SOLAS integrative studies illustrate the full meaning of interactions, paving the way for even tighter connections between thematic fields. Ultimately, SOLAS research will also develop with an enhanced consideration of societal demand while preserving fundamental research coherency. The exchange of energy, gases and particles across the air-sea interface is controlled by a variety of biological, chemical and physical processes that operate across broad spatial and temporal scales. These processes influence the composition, biogeochemical and chemical properties of both the oceanic and atmospheric boundary layers and ultimately shape the Earth system response to climate and environmental change, as detailed in the previous four chapters. In this cross-cutting chapter we present some of the SOLAS achievements over the last decade in terms of integration, upscaling observational information from process-oriented studies and expeditionary research with key tools such as remote sensing and modelling. Here we do not pretend to encompass the entire legacy of SOLAS efforts but rather offer a selective view of some of the major integrative SOLAS studies that combined available pieces of the immense jigsaw puzzle. These include, for instance, COST efforts to build up global climatologies of SOLAS relevant parameters such as dimethyl sulphide, interconnection between volcanic ash and ecosystem response in the eastern subarctic North Pacific, optimal strategy to derive basin-scale CO2 uptake with good precision, or significant reduction of the uncertainties in sea-salt aerosol source functions. Predicting the future trajectory of Earth’s climate and habitability is the main task ahead. Some possible routes for the SOLAS scientific community to reach this overarching goal conclude the chapter

Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration

Bacterial nitrous oxide (N(2)O) respiration depends on the polytopic membrane protein NosR for the expression of N(2)O reductase from the nosZ gene. We constructed His-tagged NosR and purified it from detergent-solubilized membranes of Pseudomonas stutzeri ATCC 14405. NosR is an iron-sulfur flavoprotein with redox centers positioned at opposite sides of the cytoplasmic membrane. The flavin cofactor is presumably bound covalently to an invariant threonine residue of the periplasmic domain. NosR also features conserved CX(3)CP motifs, located C-terminally of the transmembrane helices TM4 and TM6. We genetically manipulated nosR with respect to these different domains and putative functional centers and expressed recombinant derivatives in a nosR null mutant, MK418nosR::Tn5. NosR's function was studied by its effects on N(2)O respiration, NosZ synthesis, and the properties of purified NosZ proteins. Although all recombinant NosR proteins allowed the synthesis of NosZ, a loss of N(2)O respiration was observed upon deletion of most of the periplasmic domain or of the C-terminal parts beyond TM2 or upon modification of the cysteine residues in a highly conserved motif, CGWLCP, following TM4. Nonetheless, NosZ purified from the recombinant NosR background exhibited in vitro catalytic activity. Certain NosR derivatives caused an increase in NosZ of the spectral contribution from a modified catalytic Cu site. In addition to its role in nosZ expression, NosR supports in vivo N(2)O respiration. We also discuss its putative functions in electron donation and redox activation