EPR and ENDOR characterization of the reactive intermediates in the generation of NO by cryoreduced oxy-nitric oxide synthase from Geobacillus stearothermophilus.
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.
Davydov
Roman
R
Chemistry Department, Northwestern University, Evanston, Illinois 60208-3113, USA.
Sudhamsu
Jawahar
J
Lees
Nicholas S
NS
Crane
Brian R
BR
Hoffman
Brian M
BM
D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D002956:Citrulline / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009569:Nitric Oxide / Q000096:biosynthesis* / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction
D001426:Bacterial Proteins; D005296:Ferrous Compounds; D002956:Citrulline; D009569:Nitric Oxide; D006418:Heme; C068309:N(omega)-hydroxyarginine; D001120:Arginine; D019001:Nitric Oxide Synthase