Studies of the heme environment and electron flow in the inducible murine macrophage nitric oxide synthase.

Abstract

Nitric oxide synthases (NOS) catalyze the conversion of L-arginine to citrulline and nitric oxide ($\cdot$NO). Synthesis of $\cdot$NO in mammalian systems is crucial in several physiological functions, including vasodilatation, neuronal signaling, and the host-immune response. The oxidation of L-arginine utilizes NADPH and molecular oxygen. The studies described here were carried out with NOS isolated from immunostimulated murine macrophages. The enzyme has several tightly associated cofactors. There is a 6-(R)-tetrahydro-L-biopterin bound to NOS which is critical for full activity. Initial studies showed that the macrophage NOS contains one FMN and one FAD per subunit. The flavins are believed to bind in a region of NOS that has significant sequence homology to cytochrome P-450 reductase. Cytochrome P-450 reductase uses FAD and FMN to transfer the reducing equivalents of NADPH to the heme of the P-450. NOS also contains a P-450-type heme, and the same type of electron transfer has been shown to occur in NOS. The heme of NOS provides a convenient chromophore to study ligand interaction. Difference spectroscopy showed that NOS bound arginine and the intermediate, N$\sp{\rm G}$-hydroxy-L-arginine, in a similar fashion. The K$\sb{\rm d}$ for the ligands was determined to be $\sim$13 $\mu$M and $\sim$3 $\mu$M, respectively. The absence of pterin significantly increased the K$\sb{\rm d}$ for both compounds to $\sim$0.9 mM. Furthermore, a critical interrelationship between the heme and the binding of pterin was established. The path of electron flow from NADPH was investigated by stopped-flow spectroscopy. The electrons from NADPH were shown to be transferred to the flavins and then to the heme. Heme reduction was monitored by the formation of the ferrous-CO complex ($\lambda\sb{\rm max}$ = 446 nm). The reduction was biphasic, with the fast rate ranging from 0.6-3 s$\sp{-1}$ and the slow rate ranging from 0.01-0.2 s$\sp{-1}$. Integrating results from the ligand binding studies, the biphasic kinetics were explained by the existence of two populations of NOS: a fast reducing form that can bind pterin and a slow reducing form that cannot.