Premium
Pseudomonas stutzeri soluble nitrate reductase αβ‐subunit is a soluble enzyme with a similar electronic structure at the active site as the inner membrane‐bound αβγ holoenzyme
Author(s) -
Hettmann Thomas,
Anemüller Stefan,
Borcherding Heike,
Mathé Lutz,
Steinrücke Peter,
Diekmann Stephan
Publication year - 2003
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(02)03837-1
Subject(s) - pseudomonas stutzeri , protein subunit , nitrate reductase , enzyme , chemistry , biochemistry , gamma subunit , electron paramagnetic resonance , biology , bacteria , genetics , physics , nuclear magnetic resonance , gene
A two‐subunit (αβ) form of dissimilatory nitrate reductase from Pseudomonas stutzeri strain ZoBell was separated from the membrane‐residing γ‐subunit by a heat solubilization step. Here we present an optimized purification protocol leading to a soluble αβ form with high specific activity (70 U/mg). The soluble form has the stoichiometry α 1 β 1 consisting of the 130 kDa α‐subunit and the 58 kDa β‐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 αβ form with the αβγ 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 α‐ and β‐subunit to the γ‐subunit are discussed.