hsp90 is required for heme binding and activation of apo-neuronal nitric-oxide synthase: Geldanamycin-mediated oxidant generation is unrelated to any action of hsp90

Scott S. Billecke, Andrew T. Bender, Kimon C. Kanelakis, Patrick J M Murphy, Ezra R. Lowe, Yasuhiko Kamada, William B. Pratt, Yoichi Osawa

Research output: Contribution to journalArticle

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Abstract

It is established that neuronal NO synthase (nNOS) is associated with the chaperone hsp90, although the functional role for this interaction has not been defined. We have discovered that inhibition of hsp90 by radicicol or geldanamycin nearly prevents the heme-mediated activation and assembly of heme-deficient apo-nNOS in insect cells. This effect is concentration-dependent with over 75% inhibition achieved at 20 μM radicicol. The ferrous carbonyl complex of nNOS is not formed when hsp90 is inhibited, indicating that functional heme in. sertion is prevented. We propose that the hsp90-based chaperone machinery facilitates functional heme entry into apo-nNOS by the opening of the hydrophobic heme-binding cleft in the protein. Previously, it has been reported that the hsp90 inhibitor geldanamycin uncouples endothelial NOS activity and increases endothelial NOS-dependent O2-production. Geldanamycin is an ansamycin benzoquinone, and we show here that it causes oxidant production from nNOS in insect cells as well as with the purified protein. At a concentration of 20 μM, geldanamycin causes a 3-fold increase in NADPH oxidation and hydrogen peroxide formation from purified nNOS, whereas the non-quinone hsp90 inhibitor radicicol had no effect. Thus, consistent with the known propensity of other quinones, geldanamycin directly redox cycles with nNOS by a process independent of any action on hsp90, cautioning against the use of geldanamycin as a specific inhibitor of hsp90 in redox-active systems.

Original languageEnglish
Pages (from-to)20504-20509
Number of pages6
JournalJournal of Biological Chemistry
Volume277
Issue number23
DOIs
Publication statusPublished - Jun 7 2002
Externally publishedYes

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Nitric Oxide Synthase Type I
Heme
Oxidants
Nitric Oxide Synthase
Chemical activation
Oxidation-Reduction
Insects
Rifabutin
Quinones
geldanamycin
NADP
Hydrogen Peroxide
Machinery
Proteins
Oxidation
monorden

ASJC Scopus subject areas

  • Biochemistry

Cite this

hsp90 is required for heme binding and activation of apo-neuronal nitric-oxide synthase : Geldanamycin-mediated oxidant generation is unrelated to any action of hsp90. / Billecke, Scott S.; Bender, Andrew T.; Kanelakis, Kimon C.; Murphy, Patrick J M; Lowe, Ezra R.; Kamada, Yasuhiko; Pratt, William B.; Osawa, Yoichi.

In: Journal of Biological Chemistry, Vol. 277, No. 23, 07.06.2002, p. 20504-20509.

Research output: Contribution to journalArticle

Billecke, Scott S. ; Bender, Andrew T. ; Kanelakis, Kimon C. ; Murphy, Patrick J M ; Lowe, Ezra R. ; Kamada, Yasuhiko ; Pratt, William B. ; Osawa, Yoichi. / hsp90 is required for heme binding and activation of apo-neuronal nitric-oxide synthase : Geldanamycin-mediated oxidant generation is unrelated to any action of hsp90. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 23. pp. 20504-20509.
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AU - Kanelakis, Kimon C.

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