Catalytic Reduction of a Tetrahydrobiopterin Radical within Nitric-oxide Synthase

Abstract

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.

Resveratrol Reverses Endothelial Nitric-Oxide Synthase Uncoupling in Apolipoprotein E Knockout Mice

“…Among these, the NOS cofactor tetrahydrobiopterin (BH 4 ) is likely to act as the major "coupling switch" (Wei et al, 2008), and BH 4 deficiency seems to be the primary cause for eNOS uncoupling in pathophysiology (Thum et al, 2007;Li and Förstermann, 2009a). In animal models of diabetes, angiotensin II-induced hypertension and nitrate tolerance, and in spontaneously hypertensive rats, we showed that eNOS uncoupling was associated with NADPH oxidase-mediated oxidative stress.…”

Surface Charges and Regulation of FMN to Heme Electron Transfer in Nitric-oxide Synthase

“…S1) and thereby could promote a specific docking of the negatively charged FMN domain for electron transfer. Indeed, placing together the two most important residues for nNOS heme reduction (Glu 762 from the FMN domain and Lys 423 of NOSoxy) was found in our previous modeling to position the bound FMN and heme groups within a reasonable distance for electron transfer (52). Additional alignment could presumably be achieved through concerted electrostatic interactions of Lys 620 and Lys 660 .…”

Role of Increased Guanosine Triphosphate Cyclohydrolase-1 Expression and Tetrahydrobiopterin Levels upon T Cell Activation

“…The ferrous heme readily binds oxygen, forming a ferrous-dioxy (FeII-O 2 ) intermediate. BH 4 , which is also bound in the oxygenase domain, donates an electron that causes scission of FeII-O 2 to an iron-oxy species (8), which in turn hydroxylates one of the guanidino nitrogens of L-arginine, initially to N-hydroxy-L-arginine and subsequently to citrulline and NO (9,10). BH 4 also stabilizes dimers of some of NOS isoforms (11,12).…”