College of Pharmacy, University of New Mexico, Albuquerque, NM 87131, USA.
FEBS Lett. 2010 Oct 22;584(20):4335-8. doi: 10.1016/j.febslet.2010.09.028. Epub 2010 Sep 23.
The FMN-heme intraprotein electron transfer (IET) kinetics in a human inducible NOS (iNOS) oxygenase/FMN (oxyFMN) construct co-expressed with NCaM, a truncated calmodulin (CaM) construct that includes only its N-terminal globular domain consisting of residues 1-75, were determined by laser flash photolysis. The IET rate constant is significantly decreased by nearly fourfold (compared to the iNOS oxyFMN co-expressed with full length CaM). This supports an important role of full length CaM in proper interdomain FMN/heme alignment in iNOS. The IET process was not observed with added excess EDTA, suggesting that Ca(2+) depletion results in the FMN domain moving away from the heme domain. The results indicate that a Ca(2+)-dependent reorganization of the truncated CaM construct could cause a major modification of the NCaM/iNOS association resulting in a loss of the IET.
通过激光闪光光解法测定了与 NCaM（一种仅包含其包含残基 1-75 的 N 端球形结构域的截短型钙调蛋白（CaM）构建体）共表达的人诱导型 NOS（iNOS）加氧酶/FMN（oxyFMN）构建体中 FMN-血红素内蛋白电子转移（IET）的动力学。与与全长 CaM 共表达的 iNOS oxyFMN 相比，IET 速率常数显著降低了近四倍。这支持了全长 CaM 在 iNOS 中正确的结构域间 FMN/血红素排列中的重要作用。加入过量 EDTA 时未观察到 IET 过程，表明 Ca(2+)耗竭导致 FMN 结构域远离血红素结构域。结果表明，截短型 CaM 构建体的 Ca(2+)依赖性重排可能导致 NCaM/iNOS 结合的重大修饰，从而导致 IET 的丧失。
