REDUCTION OF FERRYLMYOGLOBIN AND FERRYLHEMOGLOBIN BY NITRIC-OXIDE - APROTECTIVE MECHANISM AGAINST FERRYL HEMOPROTEIN-INDUCED OXIDATIONS

The reactions of metmyoglobin (metMb) and methemoglobin (metHb), oxidi zed to their respective oxoferryl free radical species ((.)Mb-Fe-IV=O/ (.)Hb-4Fe(IV)=O) by tert-butyl hydroperoxide (t-BuOOH), with nitric ox ide (NO.) were studied by a combination of optical, electron spin reso nance (ESR), ionspray mass (MS), fluorescence, and chemiluminescence s pectrometries to gain insight into the mechanism by which NO. protects against oxidative injury produced by (.)Mb-Fe-IV=O/(.)Hb-4Fe(IV)=O. O xidation of metMb/metHb by t-BuOOH in a nitrogen atmosphere proceeded via the formation of two protein electrophilic centers, which were hem e oxoferryl and the apoprotein radical centered at tyrosine (for the ( .)Mb-Fe-IV=O form, the g value was calculated to be 2.0057), and was a ccompanied by the formation of t-BuOOH-derived tert-butyl(per)oxyl rad icals. We hypothesized that NO. may reduce both oxoferryl and apoprote in free radical electrophilic centers of (.)Mb-Fe-IV=O/(.)Hb-4Fe(IV)=O and eliminate tert-butyl(per)oxyl radicals, thus protecting against o xidative damage. We found that NO. reduced (.)Mb-Fe-IV=O/(.)Hb-4Fe(IV) =O to their respective ferric (met) forms and prevented the following: (i) oxidation of cis-parinaric acid (PnA) in liposomes, (ii) oxidatio n of luminol, and (iii) formation of the tert-butyl(per)oxyl adduct wi th the spin trap DMPO. NO. eliminated the signals of tyrosyl radical d etected by ESR and oxoferryl detected by MS in the reaction of t-BuOOH with metMb. As evidenced by MS of apomyoglobin, this effect was due t o the two-electron reduction of (.)Mb-Fe-IV=O by NO. at the oxoferryl center rather than to nitrosylation of the tyrosine residues. Results of our in vitro experiments suggest that NO. exhibits a patent, target able antioxidant effect against oxidative damage produced by oxoferryl Mb/Hb.