Identification of nitric oxide synthase as a thiolate-ligated heme protein using magnetic circular dichroism spectroscopy: Comparison with cytochrome P-450-CAM and chloroperoxidase

Masanori Sono, Dennis J. Stuehr, Masao Ikeda-Saito, John H. Dawson

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86 Citations (Scopus)

Abstract

Nitric oxide (NO) has recently been recognized as an important biomolecule playing diverse physiological roles. It is synthesized in several different tissues from L-Arg and O2, using NADPH as an electron donor, by a family of heme-containing catalytically self-sufficient monooxygenases known as nitric oxide synthases (NOS). Recently, the CO complex of reduced NOS has been shown to exhibit an absorption maximum near 450 nm, a characteristic spectral feature of cytochrome P-450 (P-450). Yet, the amino acid sequences of NOS and P-450 have no homology. To further probe the active site heme coordination structure and the heme environment of NOS, we have employed magnetic circular dichroism (MCD) and CD spectroscopy in the present study. MCD spectra of several derivatives of rat brain neuronal NOS strikingly resemble those of analogous derivatives of bacterial P-450-CAM and fungal chloroperoxidase, two known thiolate-ligated heme proteins. Given the proven fingerprinting capability of MCD spectroscopy, this provides convincing evidence for endogenous thiolate (cysteinate) ligation to the heme iron of NOS. Furthermore, the heme-related Soret CD bands of NOS (positive) and P-450s (negative), as represented by P-450-CAM, are almost mirror images, whereas chloroperoxidase exhibits totally different CD band shapes. This suggests that the active sites of NOS and P-450 may share some common structural features, but significant distinctions exist between their heme environments in certain aspects such as hydrophobicity or size.

Original languageEnglish
Pages (from-to)19943-19948
Number of pages6
JournalJournal of Biological Chemistry
Volume270
Issue number34
DOIs
Publication statusPublished - 1995 Aug 25

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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