Key result
Mutating Cys 194 in mNOS abolishes heme binding, confirming it as the proximal iron ligand.
Why the study?
The role of cysteine-194 as the proximal iron ligand in mouse macrophage nitric oxide synthase was previously suggested but lacked experimental evidence.
Experimental evidence confirms that Cys 194 is the proximal iron ligand of mouse macrophage NO-synthase.
Supports Cys194 as proximal heme ligand in murine iNOS; leaves open conservation and therapeutic relevance in human NO pathways.
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.
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Sari et al. (1996) studied this question. Mutation of Cys 194 to serine or histidine in mNOS vs. Wild type mNOS was evaluated on Catalytic oxidation of L-arginine to citrulline and heme binding. Mutation of Cys 194 to serine or histidine in mouse macrophage NO-synthase abolished L-arginine oxidation and heme binding, providing evidence that Cys 194 is the proximal iron ligand.
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