Nitric-oxide synthase (NOS) is a fusion protein composed of an oxygenase domain with a heme-active site and a reductase domain with an NADPH binding site and requires Ca 2؉ /calmodulin (CaM) for NO formation activity. We studied NO formation activity in reconstituted systems consisting of the isolated oxygenase and reductase domains of neuronal NOS with and without the CaM binding site. Reductase domains with 33-amino acid C-terminal truncations were also examined. These were shown to have faster cytochrome c reduction rates in the absence of CaM. N G -hydroxy-L-Arg, an intermediate in the physiological NO synthesis reaction, was found to be a viable substrate. Turnover rates for N Ghydroxy-L-Arg in the absence of Ca 2؉ /CaM in most of the reconstituted systems were 2.3-3.1 min ؊1 . Surprisingly, the NO formation activities with CaM binding sites on either reductase or oxygenase domains were decreased dramatically on addition of Ca 2؉ /CaM. However, NADPH oxidation and cytochrome c reduction rates were increased by the same procedure. Activation of the reductase domains by CaM addition or by C-terminal deletion failed to increase the rate of NO synthesis. Therefore, both mechanisms appear to be less important than the domain-domain interaction, which is controlled by CaM binding in wild-type neuronal NOS, but not in the reconstituted systems. The well known fusion protein, cytochrome P450BM3, is composed of a P450 oxygenase domain and a CPR domain and is also driven via intramolecular electron transfer. However, NOS is reported to have a unique intermolecular/intersubunit electron transfer system in which electrons from the reductase domain of one subunit transfer crosswise to the oxygenase domain of the other subunit in the homodimeric enzyme (9 -12). The crucial role of (6R)-5,6,7,8-tetrahydro-L-biopterin (H4B) in catalysis with NOS, probably associated with redox function and/or electron transfer, is also noted (13-15).The oxygenase domain of NOS can be efficiently expressed heterogeneously in Escherichia coli (Refs. 1-15 and references therein). It is stable, easily handled, and purified as a homodimer in the presence of H4B. However, for simplicity, this has not usually included the CaM binding site. Therefore, to further examine the role of CaM binding in catalysis, particularly its effect on the oxygenase domain, it was thought necessary to examine the catalytic properties of an oxygenase domain mutant including the CaM binding site. Conversely, most of the isolated reductase domains so far studied include the CaM-binding site to study the effect of CaM on the intramolecular electron transfer from FAD to . Intriguingly, the C termini of the reductase domains of inducible and constitute full-length NOSs to attenuate electron flow through the flavine and heme domains (19,20). The isolated reductase domain without the CaM-binding site and isolated C-terminal truncated reductase domains are also examined in this paper.* This work was supported in part by Japan Society for the Promotion of Science Fellowship 9...

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