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 α1β1 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 KM 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.
Research Areas and Centers
- Academic Focus: Center for Infection and Inflammation Research (ZIEL)