Bengea Simona, Araki Yasuyuki, Ito Osamu, Igarashi Jotaro, Sagami Ikuko, Shimizu Toru
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan.
J Inorg Biochem. 2004 Jul;98(7):1210-6. doi: 10.1016/j.jinorgbio.2004.04.009.
The effects of substrates, inhibitors and tetrahydrobiopterin (H4B) on CO rebinding to the isolated heme-bound oxygenase domain (nNOSox) of neuronal nitric oxide synthase were examined by laser flash photolysis. The rate constant of CO recombination with substrate and inhibitor-free nNOSox in the absence of H4B was 1.0 x 10(6) M(-1) s(-1). The addition of H4B led to a marked decrease in the rate to 0.59 x 10(6) M(-1) s(-1). Interestingly, the substrates, L-Arg and N-hydroxy-L-Arg (NHA), altered CO binding behavior in that the binding rate was modified to CO concentration-independent, both with and without H4B. In the absence of H4B, agmatine, NG-monomethyl-L-Arg (NMMA) and NG-nitro-L-Arg methyl ester (NAME) decreased the CO concentration-dependent rate constants of rebinding by half (0.43 x 10(6) M(-1) s(-1) for the NMMA-bound complex), whereas N6-(l-iminoethyl)-L-Lys (NIL) and 7-nitro-1H-indazole (7-NI) increased the rate constants by more than 70% (up to 2.1 x 10(6) M(-1) s(-1) for the NIL-bound complex). In the presence of H4B, the binding rate was independent of CO concentration for the agmatine-bound complex. The differential effects of the inhibitors on the CO concentration-dependent rate constants were significantly diminished for the H4B-bound system. Interestingly, these variable effects of inhibitors on the CO binding rate were more pronounced in the absence of H4B. Accordingly, we suggest that H4B significantly influences CO binding by altering the CO access channel, and further reduces the divergent effects of different inhibitors.
通过激光闪光光解研究了底物、抑制剂和四氢生物蝶呤（H4B）对一氧化碳与神经元型一氧化氮合酶分离的血红素结合加氧酶结构域（nNOSox）重新结合的影响。在没有H4B的情况下，一氧化碳与无底物和抑制剂的nNOSox重新结合的速率常数为1.0×10⁶ M⁻¹ s⁻¹。加入H4B后，速率显著降低至0.59×10⁶ M⁻¹ s⁻¹。有趣的是，底物L-精氨酸（L-Arg）和N-羟基-L-精氨酸（NHA）改变了一氧化碳的结合行为，即无论有无H4B，结合速率均变为与一氧化碳浓度无关。在没有H4B的情况下，胍丁胺、NG-甲基-L-精氨酸（NMMA）和NG-硝基-L-精氨酸甲酯（NAME）使一氧化碳浓度依赖性重新结合的速率常数降低一半（NMMA结合复合物为0.43×10⁶ M⁻¹ s⁻¹），而N⁶-（1-亚氨基乙基）-L-赖氨酸（NIL）和7-硝基-1H-吲唑（7-NI）使速率常数增加70%以上（NIL结合复合物高达2.1×10⁶ M⁻¹ s⁻¹）。在有H4B的情况下，胍丁胺结合复合物的结合速率与一氧化碳浓度无关。对于H4B结合系统，抑制剂对一氧化碳浓度依赖性速率常数的差异效应显著减小。有趣的是，在没有H4B的情况下，抑制剂对一氧化碳结合速率的这些可变效应更为明显。因此，我们认为H4B通过改变一氧化碳进入通道显著影响一氧化碳结合，并进一步降低不同抑制剂的发散效应。
