Reaction of neuronal nitric oxide synthase with the nitric oxide spin-trapping agent, iron complexed withN-dithiocarboxysarcosine

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Abstract
A water‐soluble iron complex with N‐dithiocarboxysarcosine (Fe–DTCS) has been developed as an ESR spin‐trapping agent for NO and successfully applied to ESR imaging of endogenous NO production in mice. We attempted to measure NO produced by purified neuronal NO synthase (nNOS) by this method, but could not detect NO. We speculated that Fe–DTCS inhibits NOS activity. In fact, it markedly inhibited NOS activity with an IC50 value of 9.7 ± 0.7 µm in the citrulline‐formation assay. DTCS alone did not inhibit the activity. An iron complex with N‐methyl‐d‐glucamine dithiocarbamate, a similar spin‐trapping agent for NO, also inhibited the activity, with an IC50 value of 25.1 ± 2.9 µm. Fe–DTCS suppressed cytochrome c and ferricyanide reductase activities of nNOS, and markedly increased nNOS‐mediated NADPH oxidation. Concomitantly, it accelerated oxygen consumption caused by activated nNOS. These results suggest that the ESR spin‐trapping agent Fe–DTCS inhibits NO synthesis by interfering with the physiological electron flow from NADPH to nNOS heme iron.