Feng Changjian, Tollin Gordon, Hazzard James T, Nahm Nickolas J, Guillemette J Guy, Salerno John C, Ghosh Dipak K
College of Pharmacy, University of New Mexico, Albuquerque, New Mexico 87131, USA.
J Am Chem Soc. 2007 May 2;129(17):5621-9. doi: 10.1021/ja068685b. Epub 2007 Apr 11.
Intraprotein interdomain electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in nitric oxide (NO) synthesis by NO synthase (NOS). Our previous laser flash photolysis studies have provided a direct determination of the kinetics of IET between the FMN and heme domains in truncated oxyFMN constructs of rat neuronal NOS (nNOS) and murine inducible NOS (iNOS), in which only the oxygenase and FMN domains along with the calmodulin (CaM) binding site are present [Feng, C. J.; Tollin, G.; Holliday, M. A.; Thomas, C.; Salerno, J. C.; Enemark, J. H.; Ghosh, D. K. Biochemistry 2006, 45, 6354-6362. Feng, C. J.; Thomas, C.; Holliday, M. A.; Tollin, G.; Salerno, J. C.; Ghosh, D. K.; Enemark, J. H. J. Am. Chem. Soc. 2006, 128, 3808-3811]. Here, we report the kinetics of IET between the FMN and heme domains in a rat nNOS holoenzyme in the presence and absence of added CaM using laser flash photolysis of CO dissociation in comparative studies on partially reduced NOS and a single domain NOS oxygenase construct. The IET rate constant in the presence of CaM is 36 s-1, whereas no IET was observed in the absence of CaM. The kinetics reported here are about an order of magnitude slower than the kinetics in a rat nNOS oxyFMN construct with added CaM (262 s-1). We attribute the slower IET between FMN and heme in the holoenzyme to the additional step of dissociation of the FMN domain from the reductase complex before reassociation with the oxygenase domain to form the electron-transfer competent output state complex. This work provides the first direct measurement of CaM-controlled electron transfer between catalytically significant redox couples of FMN and heme in a nNOS holoenzyme.
在一氧化氮合酶（NOS）催化合成一氧化氮（NO）的过程中，黄素单核苷酸（FMN）至血红素的蛋白质内结构域间电子转移（IET）至关重要。我们之前的激光闪光光解研究直接测定了大鼠神经元型一氧化氮合酶（nNOS）和小鼠诱导型一氧化氮合酶（iNOS）的截短氧合FMN构建体中FMN与血红素结构域之间的IET动力学，这些构建体中仅存在加氧酶和FMN结构域以及钙调蛋白（CaM）结合位点[Feng, C. J.; Tollin, G.; Holliday, M. A.; Thomas, C.; Salerno, J. C.; Enemark, J. H.; Ghosh, D. K. Biochemistry 2006, 45, 6354 - 6362. Feng, C. J.; Thomas, C.; Holliday, M. A.; Tollin, G.; Salerno, J. C.; Ghosh, D. K.; Enemark, J. H. J. Am. Chem. Soc. 2006, 128, 3808 - 3811]。在此，我们通过在部分还原的NOS和单结构域NOS加氧酶构建体的对比研究中对CO解离进行激光闪光光解，报告了在添加和未添加CaM的情况下大鼠nNOS全酶中FMN与血红素结构域之间的IET动力学。存在CaM时IET速率常数为36 s⁻¹，而在不存在CaM时未观察到IET。此处报告的动力学比添加CaM的大鼠nNOS氧合FMN构建体中的动力学（262 s⁻¹）慢约一个数量级。我们将全酶中FMN与血红素之间较慢的IET归因于FMN结构域从还原酶复合物解离的额外步骤，之后再与加氧酶结构域重新结合以形成具有电子转移能力的输出状态复合物。这项工作首次直接测量了nNOS全酶中CaM控制的FMN和血红素这两个具有催化意义的氧化还原对之间的电子转移。
