EPR and ENDOR Characterization of the Reactive Intermediates in the Generation of NO by Cryoreduced Oxy-Nitric Oxide Synthase from Geobacillus stearothermophilus

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Abstract

Cryoreduction EPR/ENDOR/step-annealing measurements with substrate complexes of oxy-gsNOS (3; gsNOS is nitric oxide synthase from Geobacillus stearothermophilus) confirm that Compound I (6) is the reactive heme species that carries out the gsNOS-catalyzed (Stage I) oxidation of L-arginine to N-hydroxy-L-arginine (NOHA), whereas the active species in the (Stage II) oxidation of NOHA to citrulline and HNO/NO(-) is the hydroperoxy-ferric form (5). When 3 is reduced by tetrahydrobiopterin (BH4), instead of an externally supplied electron, the resulting BH4(+) radical oxidizes HNO/NO(-) to NO. In this report, radiolytic one-electron reduction of 3 and its complexes with Arg, Me-Arg, and NO(2)Arg was shown by EPR and (1)H and (14,15)N ENDOR spectroscopies to generate 5; in contrast, during cryoreduction of 3/NOHA, the peroxo-ferric-gsNOS intermediate (4/NOHA) was trapped. During annealing at 145 K, ENDOR shows that 5/Arg and 5/Me-Arg (but not 5/NO(2)Arg) generate a Stage I primary product species in which the OH group of the hydroxylated substrate is coordinated to Fe(III), characteristic of 6 as the active heme center. Analysis shows that hydroxylation of Arg and Me-Arg is quantitative. Annealing of 4/NOHA at 160 K converts it first to 5/NOHA and then to the Stage II primary enzymatic product. The latter contains Fe(III) coordinated by water, characteristic of 5 as the active heme center. It further contains quantitative amounts of citrulline and HNO/NO(-); the latter reacts with the ferriheme to form the NO-ferroheme upon further annealing. Stage I delivery of the first proton of catalysis to the (unobserved) 4 formed by cryoreduction of 3 involves a bound water that may convey a proton from L-Arg, while the second proton likely derives from the carboxyl side chain of Glu 248 or the heme carboxylates; the process also involves proton delivery by water(s). In the Stage II oxidation of NOHA, the proton that converts 4/NOHA to 5/NOHA likely is derived from NOHA itself, a conclusion supported by the pH invariance of the process. The present results illustrate how the substrate itself modulates the nature and reactivity of intermediates along the monooxygenase reaction pathway.

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