Catalytic Intermediates of Inducible Nitric-oxide Synthase Stabilized by the W188H Mutation

Nitric-oxide synthase (NOS) catalyzes nitric oxide (NO) synthesis via a two-step process:l-arginine (l-Arg)→N-hydroxy-l-arginine →citrulline +NO. In the active site the heme is coordinated by a thiolate ligand, which accepts aH-bond from a nearby tryptophan residue, Trp-188. Mutation of Trp-188 to histidine inmurine inducible NOS was shown to retard NO synthesis and allow for transientaccumulation of a new intermediate with a Soret maximum at 420 nm during thel-Arg hydroxylation reaction (Tejero, J., Biswas, A., Wang, Z. Q., Page, R. C.,Haque, M. M., Hemann, C., Zweier, J. L., Misra, S., and Stuehr, D. J. (2008) J.Biol. Chem. 283, 33498–33507). However, crystallographic datashowed that the mutation did not perturb the overall structure of the enzyme. Tounderstand how the proximal mutation affects the oxygen chemistry, we carried outbiophysical studies of the W188H mutant. Our stopped-flow data showed that the 420-nmintermediate was not only populated during the l-Arg reaction but also duringthe N-hydroxy-l-arginine reaction. Spectroscopic data andstructural analysis demonstrated that the 420-nm intermediate is a hydroxide-boundferric heme species that is stabilized by an out-of-plane distortion of the hememacrocycle and a cation radical centered on the tetrahydrobiopterin cofactor. Thecurrent data add important new insights into the previously proposed catalytic mechanismof NOS (Li, D., Kabir, M., Stuehr, D. J., Rousseau, D. L., and Yeh, S. R. (2007)J. Am. Chem. Soc. 129, 6943–6951).Nitric-oxide synthase (NOS) is a heme-containing flavoenzyme that synthesizes nitricoxide (NO) from l-arginine (l-Arg) in a two-step process (Scheme 1). In the first step ofthe reaction, one molecule of O₂ and two electrons from NADPH are consumedfor the conversion of l-Arg to N-hydroxy-l-arginine(NOHA).² Inthe second step of the reaction, another molecule of O₂ and an additionalelectron from NADPH are used to convert NOHA to l-citrulline and NO. Previousstudies suggest that the two steps of the reaction follow distinct mechanisms meditatedby a compound I (Cmpd I) type of ferryl intermediate and a peroxyl intermediate,respectively (1–7). These mechanisms, however,remain elusive, as none of the putative intermediates have been experimentally observedunder solution conditions, although (hydro)peroxo intermediates have been identified atcryogenic temperatures by radiolytic reduction methods (8, 9); in addition, a Cmpd I intermediate has beenobserved after peroxyacid treatment (10).Three isoforms of NOS have been identified in mammals: neuronal NOS, endothelial NOS, andinducible NOS (iNOS). Similar to the P450 class of enzymes, the heme prosthetic group inall three isoforms of NOS is coordinated by a thiolate sidechain group of an intrinsiccysteine residue in the proximal heme pocket. In P450s, the thiolate ligand forms aH-bond with a peptide NH group (11), whereas in NOSs the analogous thiolate ligand accepts a H-bond from theside chain of a conserved tryptophan residue (Trp-188 in iNOS). It is believed that theH-bonding interaction with the tryptophan residue reduces the electron donatingcapability of the thiolate ligand in NOSs, thereby modulating the oxygen chemistryoccurring in the distal heme pocket of the enzymes (1, 12–15). The mutation of the conserved tryptophan(Trp-409) in neuronal NOS to Phe or Tyr was shown to increase the rate of NO synthesisduring multiple turnover conditions by decreasing the heme reduction rate and the degreeof NO autoinhibition (15,16). Comparable mutants ofiNOS, W188F, and W188Y, could not be overexpressed as stable recombinant forms (17); however, the W188H mutantwas successfully expressed, purified, and studied (18).It was shown that the W188H mutation slowed down the l-Arg hydroxylationreaction by stabilizing a new intermediate with a Soret maximum at 420 nm, which hadnever been observed during the wild type reaction, and that the formation of the 420-nmintermediate coincides with the disappearance of the ternary complex of the enzyme andthe formation of a H₄B radical, whereas its decay was concurrent with therecovery of the resting ferric enzyme. Tejero et al. (18) postulated that the 420-nmspecies is a catalytically competent oxygen-containing intermediate, such as a Cmpd Itype of ferryl species. Regardless of the identity of the intermediate, the datademonstrated that the mutation modulates the structural properties and biochemicalreactivity of the enzyme. However, the crystallographic data of the W188H mutant of theoxygenase domain of iNOS (iNOS_oxy) revealed that its active site structure isstrikingly similar to that of the wild type enzyme (18). In particular, the side chain of His-188,like that of Trp-188 in the wild type enzyme, formed a H-bond with the thiolate ligandof the heme.Open in a separate windowTo determine how the W188H mutation modulates the oxygen chemistry of iNOS_oxywithout significantly perturbing the active site structure of the enzyme, we carried outa series of studies of the W188H mutant with optical absorption, resonance Raman, andEPR spectroscopic methods under steady-state and single turnover conditions. Wediscovered that the mutation introduced a unique out-of-plane distortion to the hememacrocycle that stabilizes the 420-nm intermediate populated during both thel-Arg and NOHA reactions and at the same time destabilizes the NO bound to theferric heme during the NOHA reaction. The results are summarized and discussed in thecontext of the previously postulated NOS mechanism (1).