| Abstract
| - The simultaneous production of superoxide and nitric oxide bystimulated human neutrophilsleads to the formation of peroxynitrite, a physiologically importantbactericidal agent. Wehave investigated two possible pathways for the inactivation ofinducible nitric oxide synthase(NOS-II) by peroxynitrite: inactivation of NOS-II through oxidationof the tightly bound cofactorcalmodulin (CaM) and direct interaction of ONOO-/ONOOHwith the NOS-II protein. Studiesof two model peptides indicated that the Ca2+-dependentbinding to CaM of a typical high-affinity sequence, melittin, significantly prevented Met oxidation inCaM by ONOO-/ONOOH.In contrast, binding of the putative CaM-binding domain of humanhepatocyte NOS-II (NOS-II509-534) to CaM only marginallyprevented the oxidation of Met residues in CaM. Whenthenative NOS-II/CaM complex was exposed to peroxynitrite, CaM was inerttoward oxidation.Nevertheless, even small amounts of peroxynitrite abolished theactivity of NOS-II throughdirect interaction with the heme. The loss of activity wasparalleled by a decrease in hemeabsorbance and a shift of the absorbance maximum from 419 to 409 nm.The presence of thecofactor tetrahydrobiopterin during peroxynitrite exposure did notprevent inactivation of theenzyme but altered the change of the heme spectrum, i.e., a shift ofλmax from 419 to 420 nmrather than to 409 nm. In conclusion, peroxynitrite inactivatesNOS-II through changes inthe heme or its environment in NOS-II rather than via oxidation of thecofactor CaM.
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