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 [lambda max = 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 microM) as well as the reduction of cytochrome C by NADPH [Km = 34 +/- 8 microM and Vm = 25 +/- 5 mumol 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 and activities toward cytochrome C reduction identical to those of wild type NOS. However, they were unable to catalyze the oxidation of L-arginine to citrulline and did not appear to bind heme (no appearance of peaks around 400 and 446 nm for the resting enzyme and its CO complex, respectively, in visible spectroscopy). These data provide the first experimental evidence in favor of previous suggestions that Cys 194 was the proximal iron ligand of mouse mNOS.
Sari
M A
MA
Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques, URA 400 CNRS, Université Paris V, France.
Booker
S
S
Jaouen
M
M
Vadon
S
S
Boucher
J I
JI
Pompon
D
D
Mansuy
D
D
D000818:Animals; D001483:Base Sequence; D002956:Citrulline / Q000378:metabolism; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D007700:Kinetics; D008024:Ligands; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013053:Spectrophotometry
D017931:DNA Primers; D004791:Enzyme Inhibitors; D008024:Ligands; D011994:Recombinant Proteins; D002956:Citrulline; D019001:Nitric Oxide Synthase; D003545:Cysteine