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

Jesús Tejero, Ashis Biswas, Zhi Qiang Wang, Richard C. Page, Mohammad Mahfuzul Haque, Craig Hemann, Jay L. Zweier, Saurav Misra, Dennis J. Stuehr

Research output: Contribution to journalArticlepeer-review

43 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)33498-33507
Number of pages10
JournalJournal of Biological Chemistry
Issue number48
StatePublished - Nov 28 2008
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology


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