Heme-assisted S-nitrosation of a proximal thiolate in a nitric oxide transport protein

Andrzej Weichsel, Estelle M. Maes, John F. Andersen, Jesus G. Valenzuela, Tatjana Kh Shokhireva, F. Ann Walker, William "Bill" Montfort

Research output: Contribution to journalArticle

114 Citations (Scopus)

Abstract

Certain bloodsucking insects deliver nitric oxide (NO) while feeding, to induce vasodilation and inhibit blood coagulation. We have expressed, characterized, and determined the crystal structure of the Cimex lectularius (bedbug) nitrophorin, the protein responsible for NO storage and delivery, to understand how the insect successfully handles this reactive molecule. Surprisingly, NO binds not only to the ferric nitrophorin heme, but it can also be stored as an S-nitroso (SNO) conjugate of the proximal heme cysteine (Cys-60) when present at higher concentrations. EPR- and UV-visible spectroscopies, and a crystallographic structure determination to 1.75-Å resolution, reveal SNO formation to proceed with reduction of the heme iron, yielding an Fe-NO complex. Stopped-flow kinetic measurements indicate that an ordered reaction mechanism takes place: initial NO binding occurs at the ferric heme and is followed by heme reduction, Cys-60 release from the heme iron, and SNO formation. Release of NO occurs through a reversal of these steps. These data provide, to our knowledge, the first view of reversible metal-assisted SNO formation in a protein and suggest a mechanism for its role in NO release from ferrous heme. This mechanism and Cimex nitrophorin structure are completely unlike those of the nitrophorins from Rhodnius prolixus, where NO protection is provided by a large conformational change that buries the heme nitrosyl complex, highlighting the remarkable evolution of proteins that assist insects in bloodfeeding.

Original languageEnglish (US)
Pages (from-to)594-599
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number3
DOIs
StatePublished - Jan 18 2005

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Nitrosation
Heme
Carrier Proteins
Nitric Oxide
Bedbugs
Insects
Iron
Insect Proteins
Rhodnius
Blood Coagulation
Vasodilation
Cysteine
Spectrum Analysis
Proteins
Metals
nitrophorin

Keywords

  • Crystal structure
  • Heme protein
  • Nitrophorin
  • S-nitroso
  • S-nitrosocysteine

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Heme-assisted S-nitrosation of a proximal thiolate in a nitric oxide transport protein. / Weichsel, Andrzej; Maes, Estelle M.; Andersen, John F.; Valenzuela, Jesus G.; Shokhireva, Tatjana Kh; Walker, F. Ann; Montfort, William "Bill".

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 102, No. 3, 18.01.2005, p. 594-599.

Research output: Contribution to journalArticle

Weichsel, Andrzej ; Maes, Estelle M. ; Andersen, John F. ; Valenzuela, Jesus G. ; Shokhireva, Tatjana Kh ; Walker, F. Ann ; Montfort, William "Bill". / Heme-assisted S-nitrosation of a proximal thiolate in a nitric oxide transport protein. In: Proceedings of the National Academy of Sciences of the United States of America. 2005 ; Vol. 102, No. 3. pp. 594-599.
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AB - Certain bloodsucking insects deliver nitric oxide (NO) while feeding, to induce vasodilation and inhibit blood coagulation. We have expressed, characterized, and determined the crystal structure of the Cimex lectularius (bedbug) nitrophorin, the protein responsible for NO storage and delivery, to understand how the insect successfully handles this reactive molecule. Surprisingly, NO binds not only to the ferric nitrophorin heme, but it can also be stored as an S-nitroso (SNO) conjugate of the proximal heme cysteine (Cys-60) when present at higher concentrations. EPR- and UV-visible spectroscopies, and a crystallographic structure determination to 1.75-Å resolution, reveal SNO formation to proceed with reduction of the heme iron, yielding an Fe-NO complex. Stopped-flow kinetic measurements indicate that an ordered reaction mechanism takes place: initial NO binding occurs at the ferric heme and is followed by heme reduction, Cys-60 release from the heme iron, and SNO formation. Release of NO occurs through a reversal of these steps. These data provide, to our knowledge, the first view of reversible metal-assisted SNO formation in a protein and suggest a mechanism for its role in NO release from ferrous heme. This mechanism and Cimex nitrophorin structure are completely unlike those of the nitrophorins from Rhodnius prolixus, where NO protection is provided by a large conformational change that buries the heme nitrosyl complex, highlighting the remarkable evolution of proteins that assist insects in bloodfeeding.

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