EPR and ENDOR Characterization of Intermediates in the Cryoreduced Oxy-Nitric Oxide Synthase Heme Domain with Bound l-Arginine or N^G-Hydroxyarginine

Abstract

Reconstitution of the endothelial nitric oxide synthase heme domain (NOS) with the catalytically noncompetent 4-aminotetrahydrobiopterin has allowed us to prepare at -40 degrees C the oxyferrous-NOS-substrate complexes of both L-arginine (Arg) and N(G)-hydroxyarginine (NOHA). We have radiolytically cryoreduced these complexes at 77 K and used EPR and ENDOR spectroscopies to characterize the initial products of reduction, as well as intermediates that arise during stepwise annealing to higher temperatures. Peroxo-ferri-NOS is the primary product of 77 K cryoreduction when either Arg or NOHA is the substrate. Proton ENDOR spectra of this state suggest that the peroxo group is H-bonded to a [guanidinium-water] network that forms because the binding of O2 to the ferroheme of NOS recruits H2O. At no stage of reaction/annealing does one observe an EPR signal from a hydroperoxo-ferri state with either substrate. Instead, peroxo-ferri-NOS-substrate complexes convert to a product-state intermediate at the extremely low temperature of 165-170 K. EPR and proton ENDOR spectra of the intermediate formed with Arg as substrate support the suggestion that the reaction involves the formation and attack of Compound I. Within the time/temperature resolution of the present experiments, samples with Arg and NOHA as substrate behave the same in the initial steps of cryoreduction/annealing, despite the different acid/base characteristics of the two substrates. This leads us to discuss the possibility that ambient-temperature catalytic conversion of both substrates is initiated by reduction of the oxy-ferroheme to the hydroperoxo-ferriheme through a coupled proton-electron transfer from a heme-pocket reductant, and that Arg may provide the stoichiometrically second proton of catalysis.

Single-turnover of Nitric-oxide Synthase in the Presence of 4-Amino-tetrahydrobiopterin

“…Using cryoreduction techniques the formation of ferric peroxy and hydroperoxy complexes has been reported (49). The same technique was recently applied successfully to eNOS oxy , which demonstrated the formation of the peroxy complex but not of its protonated counterpart (45). However, in that study 4-amino-BH4 was used as a catalytically noncompetent pteridine, without considering the possibility that proton transfer to Fe(II)⅐O 2 Ϫ , rather than electron transfer to Fe(II)⅐O 2 , is impaired in the presence of 4-amino- (50); www.pdb.org/) as a surrogate for the O 2 complex.…”

“…The fact that protonation of Fe(II)⅐O 2 Ϫ is probably the last reaction step that is shared by the cycles with Arg and NHA, offers additional support for this hypothesis, because the differences between BH4 and 4-amino-BH4 occurred with both substrates. Indeed, it has been speculated previously that BH4 might serve as a proton donor (4,35,45). This would offer an attractive explanation for the inability of 4-amino-BH4 to support catalysis, because the most stable tautomer of 4-amino-BH4 lacks the proton at N3 that is directly linked to the heme (7,35).…”

The bioinorganic chemistry of iron in oxygenases and supramolecular assemblies

“…Many of the iron-containing oxygenases, such as cytochrome P450s, mitochondrial NOS (59)(60)(61)(62)(63)(64)(65)(66)(67), and AlkB, are membrane bound. Synthetic phospholipid vesicles have presented the opportunity to construct novel supramolecular assemblies and elucidate the membrane-binding properties of biomolecules.…”

NO formation by a catalytically self-sufficient bacterial nitric oxide synthase from Sorangium cellulosum

“…While the mechanism is still under debate, one widely accepted proposal involves a tetrahedral intermediate formed via the nucleophilic attack of a Fe III -hydroperoxo species to NHA (17, 18). In both steps, the tetrahydrobiopterin cofactor is involved in electron transfer (17,19).More recently, bacterial NOSs have been reported (20-25). Unlike eukaryotic NOSs, prokaryotic NOSs do not have a reductase domain as a contiguous polypeptide (20, 21) and consequently NO detection has been mostly limited to single turnover experiments (22).…”

“…While the mechanism is still under debate, one widely accepted proposal involves a tetrahedral intermediate formed via the nucleophilic attack of a Fe III -hydroperoxo species to NHA (17, 18). In both steps, the tetrahydrobiopterin cofactor is involved in electron transfer (17,19).…”