Loss of nitrous oxide reductase in Pseudomonas aeruginosa cultured under N2O as determined by rocket immunoelectrophoresis

The mass ratio of nitrous oxide reductase to total protein in the soluble protein fraction of Pseudomonas aeruginosa P2 was highest in cells grown on nitrate, decreased in cells grown on N(2)O following the exhaustion of the initial charge of nitrate, and was nearly zero in cells exposed solely to N(2)O.     

In vivo fluorescence excitation and absorption spectra of marine phytoplankton: I. Taxonomic characteristics and responses to photoadaptation

Absorption and fluorescence excitation spectra were measuredfor batch cultures of five species of marine phytoplankton grownunder high and low light. These spectra were examined for propertiescharacteristic of taxonomic position and of photoadaptive response.While regions of absorption and excitation of chlorophyll afluorescence diagnostic of pigment composition were identifiable,photoadaptive response had greater influence on spectral variability.Although reduced growth irradiance caused changes in both theabsorption and fluorescence excitation spectra, the fluorescenceexcitation spectrum appears to be more sensitive to alterationsin the ambient light field for growth than does the absorptionspectrum. For a single species. the fluorescence excitationspectrum for a sample grown at low irradiance showed greaterstructure than that for the sample grown at a high irradiance.Under low light conditions, the excitation of chlorophyll afluorescence by accessory pigments increased relative to theexcitation by chlorophyll a itself The highest fluorescenceyields occur in the blue-green region of the spectrum, correspondingto bands of peak absorption by the accessory pigments. Changesin absorption spectra are less marked, but two features recur.First. in the blue-green region of the spectrum from -500–560nm. absorption is enhanced in the low-light cells relative tothat of the high-light cells. Second, the ratio of absorptionat 435 nm to that at 676 nm was greater for the high-light cells.Correlating changes in pigment concentrations were observed.The influence of photoadaptation on the properties of fluorescenceexcitation spectra is as great or greater than the influenceof pigment complements characteristic of specific algal taxa.     

Loss of Nitrous Oxide Reductase in Pseudomonas aeruginosa Cultured under N2O as Determined by Rocket Immunoelectrophoresis

The mass ratio of nitrous oxide reductase to total protein in the soluble protein fraction of Pseudomonas aeruginosa P2 was highest in cells grown on nitrate, decreased in cells grown on N₂O following the exhaustion of the initial charge of nitrate, and was nearly zero in cells exposed solely to N₂O.     

Extended x-ray absorption fine structure and electron paramagnetic resonance of nitrous oxide reductase from Pseudomonas aeruginosa strain P2.

The copper centers of nitrous oxide reductase from Pseudomonas aeruginosa strain P2 were studied by x-ray and electron paramagnetic resonance (EPR) spectroscopy. The enzyme is dimeric and contains four Cu atoms and about seven cysteine residues/subunit of Mr = 73,000. The extended x-ray absorption fine structure (EX-AFS) spectrum was analyzed for enzyme as isolated (oxidized or slightly reduced), enzyme exposed briefly to air, reduced enzyme, and enzyme at pH 7 after having been activated by standing at pH 10. The average Cu ligand environment in the first shell was best modeled for all forms of the enzyme by a combination of N/O and S atoms at a total coordination number between 3 and 4 and bond distances ranging from 1.96-2.03 A for Cu-N/O and 2.20-2.25 A for Cu-S. The data could be fit without using Cu-Cu interactions. Overall the results are similar to those reported for the enzyme for Pseudomonas stutzeri (Scott, R. A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086). The first derivative EPR spectra of the Cu(II) centers at 15 and 45 K were qualitatively similar among enzyme as isolated and enzyme exposed to N2O or air. These three nominally oxidized samples showed an axial signal with g perpendicular = 2.03 and g parallel = 2.15-2.16. Hyperfine structure was observed in both the g parallel and g perpendicular regions with splittings of 43 and 25 gauss, respectively. These hyperfine components are attributed to exchange coupled Cu(I)-Cu(II) S = 1/2 (half-met) centers. In the enzyme as isolated and after exposure to N2O, about 3/4 of the Cu was EPR silent, whereas after exposure to air the signal integrated to about half the Cu concentration. The EPR spectrum of enzyme activated at pH 10 but frozen at pH 7 was a composite of spectra from activated and inactive species. The activated species presented a complex set of narrow hyperfine components which may arise from contributions from more than one species of half-met center.     

PHOTOSYNTHESIS-IRRADIANCE RELATIONSHIPS IN SEA ICE MICROALGAE FROM McMURDO SOUND,ANTARCTICA1

Sea ice microalgae in McMurdo Sound, Antarctica were examined for photosynthesis-irradiance relationships and for the extent and time course of their photoadaptation to a reduction in in situ irradiance. Algae were collected from the bottom centimeter of coarse-grained congelation ice in an area free of natural snow cover. Photosynthetic rate was determined in short term (1 h) incubations at ?2° C over a range of irradiance from 0 to 286 μE·m^?2·s^?1. Assimilation numbers were consistently below 0.1 mg C·mg chl a^?1·h^?1. The I_k's³ averaged only 7 μE·m^?2·s^?1, and photosynthesis was inhibited at irradiances above 25 μE·m^?2·s^?1. Photosynthetic parameters of the ice algal community were examined over a nine day period following the addition of 4 cm of surface snow while a control area remained snow-free. A reduction of 40% in P_m^B relative to the control occurred after two days of snow cover; α, β, I_k, and I_m were not significantly altered. Low assimilation numbers and constant standing crop size, however, suggested that the algal bloom may have already reached stationary growth phase, possibly minimizing their photoadaptive response.     

Photoadaptation in Phaeocystis pouchetii advected beneath annual sea ice in McMurdo Sound, Antarctica

A bloom of the colonial microalga Phaeocystis pouchetii wasadvected from ice-free waters to beneath 1.5 m of annual fastice in East McMurdo Sound, Antarctica in late December 1984.Strategies of photoadaptation to a reduction in growth irradianceinvolved a 3- to 4-fold increase in photosynthetic efficiencyper chlorophyll a (     

Purification and characterization of nitrous oxide reductase from Pseudomonas aeruginosa strain P2

Nitrous oxide reductase, which catalyzes the reduction of N2O to N2, was purified in a largely oxidized form from Pseudomonas aeruginosa strain P2 by a simple anaerobic procedure to yield an enzyme with a peptide purity of 95-98%. For the native (dimeric) enzyme, Mr = 120,000 and for the denatured subunit, Mr = 73,000. The enzyme contained four Cu atoms/subunit, was purple in color, and exhibited a broad absorption band at 550 nm with an extinction coefficient of about 11,000 M-1 x cm-1 referenced to the dimer. It was nearly inactive as prepared but could be activated by incubation with 2-(N-cyclohexylamino)ethane sulfonate buffer, pH 10, to specific activities as high as 27 mumol of N2O x min-1 x mg-1.Km for N2O and benzyl viologen radical cation was about 2 and 4 microM, respectively, both before and after enzyme activation. Activation increased the t1/2 for turnover-dependent inactivation from about 30 s to 5-10 min. Reduction of the enzyme by dithionite was kinetically biphasic and resulted in the loss of the 550-nm band and ultimate appearance of a 670-nm band. Isoelectric focusing revealed five components with pI values from 5.2 to 5.7. The pI values did not change following activation. The copper CD spectrum of the enzyme as prepared was different from that for the activated enzyme, whereas those for the enzyme after exposure to air and the activated enzyme were similar. Because the activated enzyme is a mixture of activated and inactive species, the specific activity of the activated species must be substantially greater than the observed value. Molecular heterogeneity may also explain the decreased optical absorbance and CD amplitude that resulted from the activation process. The data overall reinforce the view that the absorption spectrum of nitrous oxide reductase is not a good predictor of absolute activity.     

SHADE ADAPTED BENTHIC DIATOMS BENEATH ANTARCTIC SEA ICE1

A dense community of shade adapted microalgae dominated by the diatom Trachyneis aspera is associated with a siliceous sponge spicule mat in McMurdo Sound, Antarctica. Diatoms at a depth of 20 to 30 m were found attached to spicule surfaces and in the interstitial water between spicules. Ambient irradiance was less than 0.6 μE · m^?2· s^?1 due to light attenuation by surface snow, sea ice, ice algae, and the water column. Photosynthesis-irradiance relationships determined by the uptake of NaH¹⁴CO₃ revealed that benthic diatoms beneath annual sea ice were light-saturated at only 11 μE·m^?2·s^?1, putting them among the most shade adapted microalgae reported. Unlike most shade adapted microalgae, however, they were not photoinhibited even at irradiances of 300 μE·m^?2·s^?1. Although in situ primary production by benthic diatoms was low, it may provide a source of fixed carbon to the abundant benthic invertebrates when phytoplankton or ice algal carbon is unavailable.