Pseudomonas stutzeri soluble nitrate reductase alphabeta-subunit is a soluble enzyme with a similar electronic structure at the active site as the inner membrane-bound alphabetagamma holoenzyme.
A two-subunit (alphabeta) form of dissimilatory nitrate reductase from Pseudomonas stutzeri strain ZoBell was separated from the membrane-residing gamma-subunit by a heat solubilization step. Here we present an optimized purification protocol leading to a soluble alphabeta form with high specific activity (70 U/mg). The soluble form has the stoichiometry alpha(1)beta(1) consisting of the 130 kDa alpha-subunit and the 58 kDa beta-subunit. We did not observe any proteolytic cleavage in the course of the heat solubilization. The enzyme is competively inhibited by azide, but not by chlorate. It exhibits a K(M) value of 3.2 mM for nitrate. We compare the enzymatic and electron paramagnetic resonance (EPR) spectroscopic properties of the alphabeta form with the alphabetagamma holoenzyme which resides in the membrane and can be prepared by detergent extraction. The nearly identical EPR spectra for the Mo(V) signal of both enzyme preparations show that the active site is unaffected by the heat step. The factors influencing the binding of the alpha- and beta-subunit to the gamma-subunit are discussed.
Hettmann
Thomas
T
Department of Molecular Biology, IMB, Beutenbergstr. 11, D-07745 Jena, Germany.
Anemüller
Stefan
S
Borcherding
Heike
H
Mathé
Lutz
L
Steinrücke
Peter
P
Diekmann
Stephan
S
D001386:Azides / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D002704:Chlorates / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D004791:Enzyme Inhibitors / Q000494:pharmacology; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D021122:Protein Subunits; D011549:Pseudomonas / Q000201:enzymology*; D020539:Sequence Analysis, Protein; D012995:Solubility
D001386:Azides; D001426:Bacterial Proteins; D002704:Chlorates; D004791:Enzyme Inhibitors; D010743:Phospholipids; D021122:Protein Subunits; D009565:Nitrate Reductases; D050901:Nitrate Reductase