Expression in Yeast and Purification of Functional Macrophage Nitric Oxide Synthase. Evidence for Cysteine-194 As Iron Proximal Ligand

Mouse macrophage NO-synthase (mNOS) was expressed in a unique yeast-based system by using a three-step procedure which allows yeast growth and NOS expression to be uncoupled. Despite cytotoxic effects related to mNOS expression, levels of catalytically active enzyme up to 0.5 mg of protein per 5 L of culture was obtained after purification. Its electrophoretic, spectroscopic [λmax = 446 nm for its Fe(II)−CO complex], and catalytic properties were similar to those previously reported for mNOS purified from macrophages. Recombinant mNOS catalyzed the NADPH-dependent oxidation of l-arginine to citrulline (Km = 7 ± 3 μM) as well as the reduction of cytochrome C by NADPH [Km = 34 ± 8 μM and Vm = 25 ± 5 μmol min-1 (mg of protein-1)]. Two mutants of mNOS in which Cys 194 was replaced with either serine or histidine were constructed and expressed in the same yeast strain at a level higher than that of the wild type protein, as they appear less toxic for the host. Both mutants exhibited electrophoretic properties ...