Reactions catalyzed by the heme domain of inducible nitric oxide synthase: evidence for the involvement of tetrahydrobiopterin in electron transfer

摘要

The heme domain (iNOS_(heme)) of inducible nitrix oxide synthase (iNOS) was expressed in Escherichia coli and purified to homogeneity. Characterization of the expressed iNOS_(heme) shows it to behave in all respects like full-length iNOS. iNOS_(heme) is isolated without bound pterin but can be readily reconstituted with (6R)-5,6,7,8-tetrahydro-L-biopterin (H_4B) or other pterins. The reactivity of pterin-bound and pterin-free iNOS_(heme) was examined, using sodium dithionite as the reductant. H_4B-bound iNOS_(heme) catalyzes both steps of the NOS reaction, hydroxylating argining to N~G-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 H_4B-bound iNOS_(heme) with dithionite also requires 2 to 2.5 electron equiv. These data together demonstrate that fully reduced H_4B-bound iNOS_(heme) 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 iNOS_(heme) or iNOS_(heme) reconstituted with a redox-inactive analogue, 6(R,S)-methyl-5-deaza-5,6,7,8-tetrahydropterin, did not form NHA under these conditions. H_4B has an integral role in NHA oxidation as well. Pterin-free iNOS_(heme) oxidizes NHA to citrulline, N~G-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 iNOS_(heme) requires only 1 to 1.5 electron equiv, indicating that both electrons for the oxidation of NHA by pterin-free iNOS_(heme) are derived from dithionite. These data provide strong evidence that H_4B is involved in electron transfer in NOS catalysis.