Structure of tetrahydrobiopterin tunes its electron transfer to the heme-dioxy intermediate in nitric oxide synthase.
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.
Wei
Chin-Chuan
CC
Department of Immunology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Wang
Zhi-Qiang
ZQ
Arvai
Andrew S
AS
Hemann
Craig
C
Hille
Russ
R
Getzoff
Elizabeth D
ED
Stuehr
Dennis J
DJ
D000818:Animals; D001120:Arginine / Q000737:chemistry; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry*; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D004579:Electron Transport; D005609:Free Radicals / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D051379:Mice; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D052247:Nitric Oxide Synthase Type II; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D011622:Pterins / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D005609:Free Radicals; D007527:Isoenzymes; D011622:Pterins; D001708:Biopterins; D006418:Heme; D001120:Arginine; C017523:6-methyltetrahydropterin; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C003402:sapropterin; D010100:Oxygen