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Stabilization of Reaction Intermediates in the Catalytic Cycle of Nitric Oxide Synthases
Author(s) -
Tejero Jesus,
Biswas Ashis,
Wang ZhiQiang,
Page Richard C.,
Haque Mohammed Mahfuzul,
Hemann Craig,
Zweier Jay L.,
Misra Saurav,
Stuehr Dennis J.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.890.1
Subject(s) - heme , chemistry , hydroxylation , stereochemistry , hemeprotein , nitric oxide , catalytic cycle , histidine , enzyme , biochemistry , organic chemistry
Nitric oxide synthases (NOS) catalyze the synthesis of nitric oxide from L‐Arginine. NOS enzymes have a tryptophan residue that forms a hydrogen bond with the heme‐thiolate and a stacking interaction with the heme. To assess the importance of this residue we replaced it by histidine in inducible NOS. 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. The crystal structure of the mutant showed that the His188 imidazole still stacked with the heme and was in position to form a 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. Formation of this species required the presence of tetrahydrobiopterin (H 4 B) and its buildup appears to be coupled with the formation of the bound H 4 B radical. The decay of the intermediate species correlates with L‐Arg hydroxylation and formation of ferric enzyme. This indicates that the W188H mutation stabilizes an intermediate heme‐oxy species whose reactivity becomes rate‐limiting for L‐Arg hydroxylation. The conserved Trp residue in NOS may facilitate NO synthesis by tuning heme‐thiolate electronegativity. Additional research to identify the intermediate will be discussed.