Engineering a P450 BM3-like red FMN semiquinone into neuronal nitric oxide synthase

摘要

We have deleted residue Gly810 from the FMN binding loop in neuronal NOS (nNOS) to give AG810 so that the shorter binding loop mimics that in cytochrome P450BM3. As expected, the FMN semiquinone has changed from its blue neutral form in the wild type to a red anionic form in the AG810 mutant. The ox/sq redox potential now is lower than the sq/hq couple. AG810 exhibits lower NO synthase activity but normal levels of cytochrome c reductase activity. However, unlike the wild type enzyme the cytochrome c reductase activity of AG810 is insensitive to calmodulin binding. These results indicate that the FMN domain in AG810 is locked in a unique conformation which is no longer sensitive to calmodulin binding and resembles the "on" output state of the calmodulin-bound wild type nNOS with respect to the cytochrome c reduction activity.