Stabilization and characterization of a heme-oxy reaction intermediate in inducible nitric-oxide synthase

Nitric-oxide synthases (NOS) are heme-thiolate enzymes that N-hydroxylate L-arginine (L-Arg) to make NO. NOS contain a unique Trp residue whose side chain stacks with the heme and hydrogen bonds with the heme thiolate. To understand its importance we substituted His for Trp¹⁸⁸ in the inducible NOS oxygenase domain (iNOSoxy) and characterized enzyme spectral, thermodynamic, structural, kinetic, and catalytic properties. The W188H mutation had relatively small effects on L-Arg binding and on enzyme heme-CO and heme-NO absorbance spectra, but increased the heme midpoint potential by 88 mV relative to wildtype iNOSoxy, indicating it decreased heme-thiolate electronegativity. The protein crystal structure showed that the His¹⁸⁸ imidazole still stacked with the heme and was positioned to hydrogen bond with the heme thiolate. Analysis of a single turnover L-Arg hydroxylation reaction revealed that a new heme species formed during the reaction. Its build up coincided kinetically with the disappearance of the enzyme heme-dioxy species and with the formation of a tetrahydrobiopterin (H₄B) radical in the enzyme, whereas its subsequent disappearance coincided with the rate of L-Arg hydroxylation and formation of ferric enzyme. We conclude: (i) W188H iNOSoxy stabilizes a heme-oxy species that forms upon reduction of the heme-dioxyspecies by H₄B. (ii) The W188H mutation hinders either the processing or reactivity of the heme-oxy species and makes these steps become rate-limiting for L-Arg hydroxylation. Thus, the conserved Trp residue in NOS may facilitate formation and/or reactivity of the ultimate hydroxylating species by tuning heme-thiolate electronegativity.