EPR and ENDOR Characterization of the Reactive Intermediates in the Generation of NO by Cryoreduced Oxy-Nitric Oxide Synthase from Geobacillus stearothermophilus
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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DOI：
10.1021/ja906133h
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发表时间：
2009-10-14
影响因子：
15
通讯作者：
Hoffman, Brian M.
中科院分区：
文献类型：
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作者：
Davydov, Roman;Sudhamsu, Jawahar;Hoffman, Brian M.
用oxy-gsNOS的底物复合物进行冷冻还原EPR/ENDOR/分步退火测量（3）; gsNOS是来自嗜热脂肪土芽孢杆菌的一氧化氮合酶）证实化合物1（6）是进行gsNOS催化的（阶段1）L-精氨酸氧化成N-羟基-L-精氨酸（NOHA）的反应性血红素物质，而NOHA氧化为瓜氨酸和HNO/NO-（第11阶段）中的活性物质是氢过氧铁形式（5）。当3被四氢生物蝶呤（BH_4）还原时，所产生的BH_4（+）自由基代替外部提供的电子将HNO/NO-氧化成NO。本文报道了3及其与Arg、Me-Arg和NO（2）Arg的复合物的辐解单电子还原反应，用EPR和H ~（-1）和N ~（-14）、N ~（-15）ENDOR光谱证实了这一结果。相反，在3/NOHA的冷冻还原过程中，过氧铁-gsNOS中间体（4/NOHA）被捕获。在145 K下退火期间，ENDOR显示5/Arg和5/Me-Arg（但不是5/NO（2）Arg）产生第I阶段主要产物物质，其中羟基化底物的OH基团与Fe（III）配位，其特征在于6作为活性血红素中心。分析表明，Arg和Me-Arg的羟基化是定量的。4/NOHA在160 K下的退火首先将其转化为5/NOHA，然后转化为阶段11的主要酶产物。后者含有Fe（III）与水配位，其特征为5作为活性血红素中心。它还含有定量的瓜氨酸和HNO/NO-;后者在进一步退火时与亚铁血红素反应形成NO-亚铁血红素。第一阶段将催化质子传递到由3的冷冻还原形成的（未观察到的）4涉及结合水，其可以从L-Arg传递质子，而第二个质子可能来自Glu 248的羧基侧链或血红素羧酸盐;该过程还涉及通过水的质子传递。在NOHA的第11阶段氧化中，将4/NOHA转化为5/NOHA的质子可能来自NOHA本身，这一结论得到了该过程pH不变性的支持。目前的结果说明了如何基板本身调节的性质和反应性的中间体沿着单加氧酶反应途径。
Cryoreduction EPR/ENDOR/step-annealing measurements with substrate complexes of oxy-gsNOS (3; gsNOS is nitric oxide synthase from Geobacillus stearothermophilus) confirm that Compound 1 (6) is the reactive heme species that carries out the gsNOS-catalyzed (Stage 1) oxidation Of L-arginine to N-hydroXy-L-arginine (NOHA), whereas the active species in the (Stage 11) 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 H-1 and N-14,N-15 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(I I I), 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 11 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 11 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 moncoxygenase reaction pathway.