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

Abstract

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 iNOSoxy, 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.Nitric oxide synthases (NOSs) 1 are flavoheme enzymes that generate nitric oxide (NO) from L-arginine (Arg) (1, 2). The overall reaction consumes 1.5 NADPH and 2 O 2 and involves two steps, the first being Arg hydroxylation to form N-hydroxy-L-Arg (NOHA), and the second being NOHA oxidation to form citrulline and NO (see Scheme 1).The NOS heme is located in a catalytic "oxygenase" domain that also contains the binding sites for Arg and the essential redox cofactor (6R)-tetrahydrobiopterin (H 4 B) (3, 4). The heme is ligated to a cysteine thiolate (5-7) and catalyzes a reductive activation of molecular oxygen in conjunction with H 4 B in each of the two steps of NO synthesis (8 -11). The NOS heme also binds self-generated NO as an intrinsic feature of catalysis (12)(13)(14). Each molecule of NO generated by NOS has a high probability of binding to the heme before it is released from the enzyme. The resulting ferric heme-NO product complex (Fe III NO) has been observed to build up as a transient species during NOHA oxidation reactions catalyzed by NOS oxygenase domains (NOSoxy) when run in a stopped-flow spectrophotometer under single turnover conditions (9,13,14). These experiments also provided spectral and kinetic information regarding the formation of an initial ferrous heme-dioxy species (Fe II O 2 ), whose disappearance then coincides with k cat and formation of the transient Fe III NO product complex (see Scheme 2). Because dissociation of the Fe III NO product complex is required for NO to escape from the enzyme, it is an essential step for the biologic functions of animal NOSs. In fact, this dissociation rate is one of three key kinetic parameters that together determine the overall catalytic behavior of a given NOS (15,16). Slower rates of NO dissociation dispose the Fe III NO product complex toward its reduction during catalysis, which then places the ferrous heme-NO enzyme species into a futile cycle that does not release NO (Fig. 1). Interestingly, there is only a modest variation in Fe III NO dissociation rates among the three mammalian NOS isoforms, which range from 2 to 5 s Ϫ1 when measured in NOHA single turnover reactions at 10°C (16).Given the above, we...

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