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Structure of tetrahydrobiopterin tunes its electron transfer to the heme-dioxy intermediate in nitric oxide synthase

Academic Article
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Overview

authors

  • Wei, C. C.
  • Wang, Z. Q.
  • Arvai, A. S.
  • Hemann, C.
  • Hille, R.
  • Getzoff, Elizabeth
  • Stuehr, D. J.

publication date

  • 2003

journal

  • Biochemistry  Journal

abstract

  • How 6R-tetrahydrobiopterin (H(4)B) participates in Arg hydroxylation as catalyzed by the nitric oxide synthases (NOSs) is a topic of current interest. Previous work with the oxygenase domain of inducible NOS (iNOSoxy) demonstrated that H(4)B radical formation is kinetically coupled to disappearance of an initial heme-dioxy intermediate and to Arg hydroxylation in a single turnover reaction run at 10 degrees C [Wei, C.-C., Wang, Z.-Q., Wang, Q., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) J. Biol. Chem. 276, 315-319]. Here we used 5-methyl-H(4)B to investigate how pterin structure influences radical formation and associated catalytic steps. In the presence of Arg, the heme-dioxy intermediate in 5-methyl-H(4)B-bound iNOSoxy reacted at a rate of 35 s(-)(1), which is 3-fold faster than with H(4)B. This was coupled to a faster rate of 5-methyl-H(4)B radical formation (40 vs 12.5 s(-)(1)) and to a faster and more productive Arg hydroxylation. The EPR spectrum of the enzyme-bound 5-methyl-H(4)B radical had different hyperfine structure than the bound H(4)B radical and exhibited a 3-fold longer half-life after its formation. A crystal structure of 5-methyl-H(4)B-bound iNOSoxy revealed that there are minimal changes in conformation of the bound pterin or in its interactions with the protein as compared to H(4)B. Together, we conclude the following: (1) The rate of heme-dioxy reduction is linked to pterin radical formation and is sensitive to pterin structure. (2) Faster heme-dioxy reduction increases the efficiency of Arg hydroxylation but still remains rate limiting for the reaction. (3) The 5-methyl group influences heme-dioxy reduction by altering the electronic properties of the pterin rather than changing protein structure or interactions. (4) Faster electron transfer from 5-methyl-H(4)B may be due to increased radical stability afforded by the N-5 methyl group.

subject areas

  • Animals
  • Arginine
  • Biopterin
  • Catalysis
  • Crystallization
  • Crystallography, X-Ray
  • Electron Transport
  • Free Radicals
  • Heme
  • Isoenzymes
  • Kinetics
  • Mice
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type II
  • Oxidation-Reduction
  • Oxygen
  • Protein Binding
  • Pterins
  • Spectrophotometry, Ultraviolet
  • Structure-Activity Relationship
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Identity

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi026898h

PubMed ID

  • 12590583
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Additional Document Info

start page

  • 1969

end page

  • 1977

volume

  • 42

issue

  • 7

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