Feng Changjian, Dupont Andrea L, Nahm Nickolas J, Spratt Donald E, Hazzard James T, Weinberg J Brice, Guillemette J Guy, Tollin Gordon, Ghosh Dipak K
College of Pharmacy, University of New Mexico, Albuquerque, NM 87131, USA.
J Biol Inorg Chem. 2009 Jan;14(1):133-42. doi: 10.1007/s00775-008-0431-2. Epub 2008 Oct 2.
Intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in NO synthesis by NO synthase (NOS). Our previous laser flash photolysis studies provided a direct determination of the kinetics of the FMN-heme IET in a truncated two-domain construct (oxyFMN) of murine inducible NOS (iNOS), in which only the oxygenase and FMN domains along with the calmodulin (CaM) binding site are present (Feng et al. J. Am. Chem. Soc. 128, 3808-3811, 2006). Here we report the kinetics of the IET in a human iNOS oxyFMN construct, a human iNOS holoenzyme, and a murine iNOS holoenzyme, using CO photolysis in comparative studies on partially reduced NOS and a NOS oxygenase construct that lacks the FMN domain. The IET rate constants for the human and murine iNOS holoenzymes are 34 +/- 5 and 35 +/- 3 s(-1), respectively, thereby providing a direct measurement of this IET between the catalytically significant redox couples of FMN and heme in the iNOS holoenzyme. These values are approximately an order of magnitude smaller than that in the corresponding iNOS oxyFMN construct, suggesting that in the holoenzyme the rate-limiting step in the IET is the conversion of the shielded electron-accepting (input) state to a new electron-donating (output) state. The fact that there is no rapid IET component in the kinetic traces obtained with the iNOS holoenzyme implies that the enzyme remains mainly in the input state. The IET rate constant value for the iNOS holoenzyme is similar to that obtained for a CaM-bound neuronal NOS holoenzyme, suggesting that CaM activation effectively removes the inhibitory effect of the unique autoregulatory insert in neuronal NOS.
在一氧化氮合酶（NOS）合成一氧化氮（NO）的过程中，黄素单核苷酸（FMN）到血红素的蛋白质内电子转移（IET）至关重要。我们之前的激光闪光光解研究直接测定了小鼠诱导型NOS（iNOS）的截短双结构域构建体（oxyFMN）中FMN - 血红素IET的动力学，该构建体仅包含加氧酶和FMN结构域以及钙调蛋白（CaM）结合位点（Feng等人，《美国化学会志》128，3808 - 3811，2006）。在此，我们报告了人iNOS oxyFMN构建体、人iNOS全酶和小鼠iNOS全酶中IET的动力学，通过CO光解对部分还原的NOS和缺乏FMN结构域的NOS加氧酶构建体进行比较研究。人iNOS和小鼠iNOS全酶的IET速率常数分别为34±5和35±3 s⁻¹，从而直接测量了iNOS全酶中FMN和血红素这两个具有催化意义的氧化还原对之间的IET。这些值比相应的iNOS oxyFMN构建体中的值小约一个数量级，这表明在全酶中IET的限速步骤是屏蔽的电子接受（输入）状态向新的电子供体（输出）状态的转变。用iNOS全酶获得的动力学曲线中没有快速IET成分这一事实意味着该酶主要保持在输入状态。iNOS全酶的IET速率常数与钙调蛋白结合的神经元NOS全酶获得的值相似，这表明钙调蛋白激活有效地消除了神经元NOS中独特的自动调节插入序列的抑制作用。
