Structure-Function Relationship of Biodeseigned NO Synthase and Dynamics of NO

(1)一氧化氮合酶(NOS)具有与细胞色素P450(P450)类似的硫氧配位的血红素活性部位。在本研究中，我们研究了在不同底物存在的情况下，细胞色素P450 1A2(P450 1A2)远端突变体没有结合。我们发现，P450 1A2远端Glu318到Ala的突变在P450反应的O_2激活中可能是重要的，显著促进了非铁复合体的还原。加入1，2：3，4-二苯并茂或菲后，对NO复合体的诱变作用几乎消失。基于这些结果，结合其他光谱和动力学数据，我们认为P450的NO铁络合物的稳定性很大程度上归因于NO与远端羧基之间的离子桥。(2)我们研究了N、G-羟基-L-精氨酸(NHA)与过氧化氢支持的分流系统以及由P450 1A2和还原酶组成的重组系统合成大鼠肝细胞色素P450 1A2(P450 1A2)的能力。研究了P4501A2远端氨基酸在催化功能中的作用。在k&lt；cat&gt；=0.6-1.2nmol/nmolP450/min的分流反应条件下，有效地合成了NO。NHA在还原酶作用下生成NO，其重组体系的周转次数分别为26和62pmol/nmolP450/min。P450 1A2的Glu318Ala突变使分流活性增加了7.3倍，而突变使重组系统的分流活性丧失。在重组系统中，过氧化氢酶显著抑制了分流活性，而分流活性却提高了2.2倍。超氧化物歧化酶和(6R)-5，6，7，8-四氢L生物蝶呤能显著促进重组系统和分流系统的NO合成，但对重组系统和分流系统的NO合成均有明显的抑制作用。

(1)Nitric oxide synthase (NOS) has a thiolate-coordinated heme active site similar to that of cytochrome P450 (P450). In the present study, NO bindings to cytochrome P450 1A2 (P450 1A2) distal mutants were studied in the presence of various substrates. We found that a mutation at Glu318 to Ala in the putative distal site of P450 1A2, suggested to be important in the O_2 activation of P450 reactions, markedly facilitates the reduction of the NO-ferric complex. Addition of 1,2 : 3,4-dibenzanthracene or phenanthrene almost abolished the mutation effect on the NO complex. Based on these results, together with other spectral and kinetics data, it is suggested that the NO-ferric complex stability of P450, and perhaps of NOS,is largely ascribed to an ionic bridge between NO and the distal carboxyl group.(2)We examined NO synthesis capability of rat liver cytochrome P450 1A2 (P450 1A2) from N^G-hydroxy-L-Arg (NHA) with both the peroxide-supported shunt system and the reconstituted system composed of P450 1A2 and the reductase. Roles of distal amino acids of P450 1A2 in the catalytic functions were also studied. No was synthesized effectively with the shunt reactions with k_<cat>=0.6-1.2nmol/nmolP450/min. NO was formed from NHA with the reductase alone, as well as, with the reconstituted system with turnover numbers of 26 and 62 pmol/nmolP450/min, respectively. A Glu318Ala mutation of P450 1A2 enhanced the shuntreaction activity up to 7.3-fold, whereas the mutation abolished the activity with the reconstituted system. Catalase markedly inhibited the activity in the reconstituted system, whereas it enhanced the shunt activity up to 2.2-fold. Superoxide dismutase and (6R)-5,6,7,8-tetrahydro-L-biopterin, which markedly enhance NO synthesis with NOS,strongly inhibited the NO synthesis in both the reconstituted and shunt systems.