Purification and characterization of a [3Fe-4S] [4Fe-4S] type ferredoxin from hyperthermophilic archaeon, Pyrobaculum islandicum

金沢大学理工研究域自然システム学系A ferredoxin was purfied from the hyperthermophilic archaeon, Pyrobaculum islandicum. EPR spectra and metal content analyses suggested that the ferredoxin molecule contained one [3Fe-4S] and one [4Fe-4S] cluster. The ferredoxin was rapidly reduced by 8-oxoglutarate: ferredoxin oxidoreductase purfied from P. islandicum, indicating that it functions physiologically as an electron sink for the redox enzymes participating in glycolytic metabolism. Furthermore, the amino acid sequence of the P. islandicum ferredoxin was compared with those of several other bacterial ferredoxins

Purification and characterization of dissimilatory nitrate reductase from a denitrifying halophilic archaeon, Haloarcula marismortui

AbstractDissimilatory nitrate reductase was purified from a denitrifying halophilic archaeon, Haloarcula marismortui, to an electrophoretically homogeneous state. The purified enzyme was inferred to be a homotetramer composed of a 63 kDa polypeptide. The electron paramagnetic resonance spectrum of the purified enzyme revealed typical rhombic signals which were ascribed to Mo(V) in the Mo–molybdopterin complex. Like the bacterial membrane-bound (Nar-) enzyme, the purified enzyme supported the catalysis of chlorate. The enzyme was activated in extreme saline conditions and the values of kcat and Km toward nitrate were 145 s−1 and 79 μM, respectively, in the presence of 2.0 M NaCl

Draft Genome Sequence of Nitrosococcus oceani Strain NS58, a Marine Ammonia-Oxidizing Gammaproteobacterium Isolated from Tokyo Bay Sediment

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Growth phenotype analysis of heme synthetic enzymes in a halophilic archaeon, Haloferax volcanii

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Anaerobic growth variant phenotype of the Δ<i>ahbD</i> variant of <i>H</i>. <i>volcanii</i>.

<p>The <i>ahbD</i> deletion variant of <i>H</i>. <i>volcanii</i>, strain A02 (open triangles), and the parent strain H26 (open squares) were cultivated under aerobic or anaerobic conditions. As shown in (<b>A</b>), strain A02 grew actively under aerobic conditions with a similar growth rate to that observed in strain H26. Under the denitrifying condition, growth of the strain A02 was strongly inhibited as shown in (<b>B</b>). Denitrifying growth of strain A02 was not recovered by adding protoheme up to 5 μM (closed triangles) using the stock solution of protoheme dissolved in the Tris-HCl buffer. The growth was restored in some extent by further addition of 1 mM DMSO to the medium (closed diamonds), the OD₆₀₀ value reached approximately 0.6. Denitrifying growth of strain H26 was not affected by addition of 5 μM protoheme (DMSO was not added, closed squares). As indicated in (<b>C</b>), with supplementation of 5 μM protoheme, anaerobic growth of the strain A02 by DMSO-respiration (closed triangles) was restored to a similar level to that of strain H26 supplemented with protoheme (closed squares). Experiments were performed independently three times. Error bars represent S.E.</p

Molecular characterization of PitA by limited proteolytic analysis.

<p>SDS-PAGE of the proteinase K-digested fragments of PitA is shown in (<b>A</b>). Numbers below the lanes indicate each reaction times (min) after the start of proteolysis. In lanes P and K, the purified PitA and proteinase K used in the experiment, respectively, were loaded. The <i>M</i>_r standard was loaded in lane S. Estimated <i>M</i>_rs of the fragments are indicated on the right side of the gel, while those of the standard proteins are on the left side. In (<b>B</b>), the proteolytic digest obtained after the 120 min reaction was fractionated by gel filtration. Proteins and heme moieties collected in each fractions were monitored by measuring the absorbance at 280 nm (closed triangles) and at 410 nm (open circles), respectively. Apparent <i>M</i>_rs of the two fractions, I and II, that were eluted at around the 100^th and 147^th tubes, respectively, were estimated as shown in (<b>C</b>) using the <i>M</i>_r standard proteins as described in the Methods. Estimation of the <i>M</i>_r of PitA in the mature state was performed similarly. Results of the SDS-PAGE of the fractions I and II are indicated in (<b>D</b>). N-terminal amino acid sequences of each fragments were determined as described in the Results and Discussion. A possible structural model of the PitA estimated from these results is represented in (<b>E</b>).</p