A Conserved Val to Ile Switch near the Heme Pocket of Animal and Bacterial Nitric-oxide Synthases Helps Determine Their Distinct Catalytic Profiles (original) (raw)

2004, Journal of Biological Chemistry

about 20-fold slower than in mammalian NOSs. Crystal structures suggest that a conserved Val to Ile switch near the heme pocket of bsNOS might determine its kinetic profile. To test this we generated complementary mutations in the mouse inducible NOS oxygenase domain (iN-OSoxy, V346I) and in bsNOS (I224V) and characterized the kinetics and extent of their NO synthesis from NOHA and their NO-binding kinetics. The mutations did not greatly alter binding of Arg, (6R)-tetrahydrobiopterin, or alter the electronic properties of the heme or various heme-ligand complexes. Stopped-flow spectroscopy was used to study heme transitions during single turnover NOHA reactions. I224V bsNOS displayed three heme transitions involving four species as typically occurs in wildtype NOS, the beginning ferrous enzyme, a ferrous-dioxy (Fe II O 2 ) intermediate, Fe III NO, and an ending ferric enzyme. The rate of each transition was increased relative to wild-type bsNOS, with Fe III NO dissociation being 3.6 times faster. In V346I iNOSoxy we consecutively observed the beginning ferrous, Fe II O 2 , a mixture of Fe III NO and ferric heme species, and ending ferric enzyme. The rate of each transition was decreased relative to wild-type iN-OSoxy, with the Fe III NO dissociation being 3 times slower. An independent measure of NO binding kinetics confirmed that V346I iNOSoxy has slower NO binding and dissociation than wild-type. Citrulline production by both mutants was only slightly lower than wild-type enzymes, indicating good coupling. Our data suggest that a greater shielding of the heme pocket caused by the Val/Ile switch slows down NO synthesis and NO release in NOS, and thus identifies a structural basis for regulating these kinetic variables.

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