Stabilization and Characterization of a Heme-Oxy Reaction Intermediate in Inducible Nitric-oxide Synthase*□S

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 impor-tancewesubstitutedHis forTrp188 inthe inducibleNOSoxygenase domain (iNOSoxy) and characterized enzyme spectral, thermody-namic, 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 wild-type iNOSoxy, indicating it decreased heme-thiolate electronega-tivity. The protein crystal structure showed that theHis188 imidaz-ole 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 forme