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A. R. Hurshman and M. A. Marletta. 2002. Reactions Catalyzed by the Heme Domain of Inducible Nitric Oxide Synthase: Evidence for the Involvement of Tetrahydrobiopterin in Electron Transfer. Biochemistry 41: 3439-3456. Full Article

The heme domain (iNOSheme) of inducible nitric oxide synthase (iNOS) was expressed in Escherichia coli and purified to homogeneity. Characterization of the expressed iNOSheme shows it to behave in all respects like full-length iNOS. iNOSheme is isolated without bound pterin but can be readily reconstituted with (6R)-5,6,7,8-tetrahydro-L-biopterin (H4B) or other pterins. The reactivity of pterin-bound and pterin-free iNOSheme was examined, using sodium dithionite as the reductant. H4B-bound iNOSheme catalyzes both steps of the NOS reaction, hydroxylating arginine to NG-hydroxy-L-arginine (NHA) and oxidizing NHA to citrulline and �NO. Maximal product formation (0.93 � 0.12 equiv of NHA from arginine and 0.83 � 0.08 equiv of citrulline from NHA) requires the addition of 2 to 2.5 electron equiv. Full reduction of H4B-bound iNOSheme with dithionite also requires 2 to 2.5 electron equiv. These data together demonstrate that fully reduced H4B-bound iNOSheme is able to catalyze the formation of 1 equiv of product in the absence of electrons from dithionite. Arginine hydroxylation requires the presence of a bound, redox-active tetrahydropterin; pterin-free iNOSheme or iNOSheme reconstituted with a redox-inactive analogue, 6(R,S)-methyl-5-deaza-5,6,7,8-tetrahydropterin, did not form NHA under these conditions. H4B has an integral role in NHA oxidation as well. Pterin-free iNOSheme oxidizes NHA to citrulline, N-cyanoornithine, an unidentified amino acid, and NO-. Maximal product formation (0.75 � 0.01 equiv of amino acid products) requires the addition of 2 to 2.5 electron equiv, but reduction of pterin-free iNOSheme requires only 1 to 1.5 electron equiv, indicating that both electrons for the oxidation of NHA by pterin-free iNOSheme are derived from dithionite. These data provide strong evidence that H4B is involved in electron transfer in NOS catalysis.

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