NADPH nitric oxide synthase binding

In 1989, BH4 was found to be a cofactor for nitric oxide synthase (NOS) [ 126, 127]. BH4 is also involved in dimerization of NOS, as NOS is catalytically active in a homodimer structure. Three isoforms of NOS exist neuronal NOS (NOS 1), inducible NOS (NOS 2) and endothelial NOS (NOS 3). BH4 is essential for all NOS isoforms. The NOS isoforms share approximately 50-60% sequence homology. Each NOS polypeptide is comprised of oxygenase and reductase domains. An N-terminal oxygenase domain contains iron protoporphyrin IX (heme), BH4 and an arginine binding site, and a C-terminal reductase domain contains flavin mononucleotide (FMN), and a reduced nicotin-amide adenine dinucleotide phosphate (NADPH) binding site. 

Figure 11.4. Reaction catalyzedby Nitric oxide synthase (a), and arrangement of the coenzymes in the dimer (b). Electrons flow from NADPH and flavins of one snbnnit to the heme of the other. Arg denotes the snbstrate binding site. Tetrahydrobiopterin (BH4) also participates in electron transfer.

Figure 2 Nitric oxide synthase, (a). Domain architecture of NOS. The heme domain binds Zn + (gray box), heme (gray parallelogram), and H4B (white box). The reductase domain binds FMN, FAD, and NADPH (white boxes). CaM (white box) is between the heme domain and the reductase domain, (b). Two-step reaction scheme for NO synthesis by NOS.

Figure 4.3. Schematic outline of the diflavin reductase family. Members contain an N-terminal FMN-binding flavodoxin-like domain and a C-terminal FAD/NADPH-binding ferredoxin reductase-like domain, which contains an additional linker region. Shown are CPR, which has an amino-terminal membrane anchor region (Anc) NRl (Novel reductase 1) MSR (methionine synthase reductase), which contains an additional inlerdomain sequence P450 BM3, which is fused to a P450 domain and NOS (nitric oxide synthases), which has linked to a heme-containing oxygenase domain that is structurally distinct from the P450s.

FIGURE I Schematic alignment of the deduced amino acid sequences of nitric oxide synthases (NOSs) and the cytochrome P-450 reductase. Depicted are consensus binding sites for heme, L-arginine, calmodulin (CaM), flavin mononucleotide (FMN), flavin-adenine dinucleotide (FAD), and NADPH. An NH2-terminal myristoylation site (myr) is present only in the endothelial constitutive NOS (ecNOS). n. Neuronal i, inducible. 

FIGURE I Role for calmodulin (CaM) in triggering interdomain electron transfer to the nitric oxide synthase (NOS) heme iron. Electrons derived from NADPH can transfer only into the flavin centers of CaM-free neuronal NOS (A). CaM binding to NOS occurs in response to elevated Ca concentrations, and this enables electrons to transfer from the flavins to the heme iron. Heme iron reduction is associated with increased NADPH oxidation and results in (B) superoxide (O2) production in the absence of L-arginine or (C) nitric oxide (NO) synthesis in the presence of L-arginine. FAD, Flavin-adenine dinucleotide FMN, flavin mononucleotide. 

FIGURE 2 Proposed dual mode for calmodulin (CaM) control of nitric oxide synthase (NOS) electron transfer. Neuronal NOS is composed of a reductase and an oxygenase domain, shown as two circles. CaM binding to NOS activates at two points in the electron transfer sequence (1) It increases the rate at which NADPH-derived electrons are transferred into the flavins, and (2) it enables the flavins to pass electrons to the oxygenase domain of NOS. Activation at the first point is associated with an increase in reductase domain-specific catalytic activities, such as electron transfer to cytochrome c or ferricyanide (FeCN ). Activation at the second point is associated with a reduction of NOS heme iron, an initiation of NO synthesis from L-arginine (Arg), or a reduction of Oj to form superoxide (O2) in the absence of substrate. FAD, Flavin-adenine dinucleotide FMN, flavin mononucleotide NO, nitric oxide. 

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