The Three Nitric-oxide Synthases Differ in Their Kinetics of Tetrahydrobiopterin Radical Formation, Heme-Dioxy Reduction, and Arginine Hydroxylation

Wei, Chin‐Chuan; Wang, Zhi Qiang; Durra, Deborah; Hemann, Craig; Hille, Russ; Garcin, Elsa D.; Getzoff, Elizabeth D.; Stuehr, Dennis J.

doi:10.1074/jbc.m409737200

Cited by 51 publications

(42 citation statements)

References 46 publications

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“…The rate of radical disappearance in the CaM-free nNOS reaction is similar to the rate of 5MeH 4 B radical oxidation in nNOSoxy (0.2 s Ϫ1 ) following a single turnover Arg hydroxylation (12). Fig.…”

supporting

confidence: 59%

“…However, Arg and 5MeH 4 B were present in our reactions at saturating concentrations and the nNOS single turnover reaction has been shown to be well coupled under this circumstance (12). Furthermore, the EPR spectrum of superoxide has a very high P1 ⁄ 2 value (ϳ50 milliwatts) and a different line shape compared with the 5MeH 4 B and FSQ radicals (23), and so should be easy to detect by our analysis.…”

Section: Resultsmentioning

confidence: 68%

“…Moreover, the rate we measured for the reduction (1.3 s Ϫ1 at 10°C) argues that it is kinetically competent, i.e. pterin radical reduction is fast enough to circumvent the slower and irreversible oxidation of the bound pterin radical, which occurs at 0.2 s Ϫ1 in nNOS at 10°C (12). The measured rates of these two opposing redox transformations now provide a kinetic rationale for H 4 B redox cycling in NOS enzymes, and thus explain how one H 4 B molecule tightly bound in NOS enables the enzyme to produce a greater than stoichiometric amount of NO (1, 2, 6 -8, 28).…”

Section: Discussionmentioning

confidence: 77%

“…The EPR spectrum of the 5MeH 4 B radical was obtained by mixing 5MeH 4 B-bound ferrous nNOSoxy with O 2 -saturated buffer and then rapid freezing after 125 ms of reaction as performed previously (12). The EPR spectral properties of the flavin semiquinone radical (FSQ) were recorded for the nNOS flavoprotein domain (20) and for the H 2 B-bound, nNOS fulllength protein (24).…”

Section: Methodsmentioning

confidence: 99%

“…1). However, the fact that bound H 4 B radical undergoes a time-dependent oxidation (10,12,13), and that NOHA can dissociate from NOS after it forms (14,15), places a time constraint on H 4 B radical reduction. Following Arg hydroxylation, the H 4 B radical remains in the ferric enzyme (Fig.…”

mentioning

confidence: 99%

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Catalytic Reduction of a Tetrahydrobiopterin Radical within Nitric-oxide Synthase

Wei

Wang

Tejero

et al. 2008

Journal of Biological Chemistry

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Nitric-oxide synthases (NOS) are catalytically self-sufficient flavo-heme enzymes that generate NO from arginine (Arg) and display a novel utilization of their tetrahydrobiopterin (H 4 B) cofactor. During Arg hydroxylation, H 4 B acts as a one-electron donor and is then presumed to redox cycle (i.e. be reduced back to H 4 B) within NOS before further catalysis can proceed. Whereas H 4 B radical formation is well characterized, the subsequent presumed radical reduction has not been demonstrated, and its potential mechanisms are unknown. We investigated radical reduction during a single turnover Arg hydroxylation reaction catalyzed by neuronal NOS to document the process, determine its kinetics, and test for involvement of the NOS flavoprotein domain. We utilized a freeze-quench instrument, the biopterin analog 5-methyl-H 4 B, and a method that could separately quantify the flavin and pterin radicals that formed in NOS during the reaction. Our results establish that the NOS flavoprotein domain catalyzes reduction of the biopterin radical following Arg hydroxylation. The reduction is calmodulin-dependent and occurs at a rate that is similar to heme reduction and fast enough to explain H 4 B redox cycling in NOS. These results, in light of existing NOS crystal structures, suggest a "throughheme" mechanism may operate for H 4 B radical reduction in NOS.

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supporting

confidence: 59%

Section: Resultsmentioning

confidence: 68%

Section: Discussionmentioning

confidence: 77%