Tetrahydrobiopterin binding to macrophage inducible nitric oxide synthase: heme spin shift and dimer stabilization by the potent pterin antagonist 4-amino-tetrahydrobiopterin.

Abstract

The characteristics of tetrahydrobiopterin (H4biopterin) binding to pteridine-free recombinant macrophage inducible nitric oxide synthase expressed in Escherichia coli were investigated with a special focus given to effects caused by 2,4-diamino-5,6,7, 8-tetrahydro-6-(l-erythro-1,2-dihydroxypropyl)pteridine (4-amino-H4biopterin), a novel pterin-based inhibitor of nitric oxide synthase. The 4-amino compound completely inhibited enzyme stimulation by 10 microM H4biopterin with a half-maximally active concentration of 7.2 +/- 0.39 microM, whereas H2biopterin and sepiapterin were much less potent. Binding studies using [3H]H4biopterin at 4 degrees C revealed biphasic association of the radioligand according to two first-order reactions with apparent rate constants of 2.2 and 0.05 min-1, each accounting for approximately 50% of total binding. Dissociation of [3H]H4biopterin occurred with rate constants of 0.005 and 0.0028 min-1 in the absence and presence of l-arginine, respectively. Specific binding of 10 nM [3H]H4biopterin was antagonized by unlabeled H4biopterin and its 4-amino analog with half-maximal effects at 84 +/- 6 and 34 +/- 3.2 nM, respectively. Binding of H4biopterin and 4-amino-H4biopterin was accompanied by a partial low spin to high spin conversion of the heme that was completed by l-arginine. Similarly, the active cofactor and the inhibitory 4-amino derivative both induced significant formation of stable protein dimers that survived during SDS electrophoresis, suggesting that the allosteric effects caused by H4biopterin do not explain sufficiently the essential role of the pteridine cofactor in NO biosynthesis.