Effect of nitric oxide on hemoprotein-catalyzed oxidative reactions

Hemoglobin or myoglobin-catalyzed oxidation reactions have been suggested to initiate and/or exacerbate tissue injury associated with a variety of pathological conditions including post-ischemic tissue injury, hemorrhagic disorders, and chronic inflammation. In the present study, we investigated what effect different fluxes of nitric oxide (NO) have on hemoprotein-catalyzed oxidation reactions in vitro. The hypoxanthine/xanthine oxidase system was used to generate both O-2(-) and H2O2, whereas the spontaneous decomposition of the spermine\NO adduct was used to generate NO at a known and constant rate. We assessed the ability of myoglobin (Mb) or hemoglobin (Hb) to oxidize dihydrorhodamine (DHR) to rhodamine (RH) in the presence of O-2(-)/H2O2 and/or NO. In the presence of a constant flux of O-2(-) and H2O2 (1 nmol/min each), 500 nM MetMb (Fe3+) stimulated DHR oxidation from normally undetectable levels to similar to 35 mu M. This oxidation reaction was inhibited by catalase but not SOD, suggesting the formation of the ferryl-hemoprotein adduct (Fe4+). Equimolar fluxes of O-2(-), H2O2, and NO increased further DHR oxidation to similar to 50 mu M. The 15 mu M increase in DHR oxidation was independent of heme concentration and was inhibited by SOD. This suggested that equal fluxes of O-2(-) and NO interact to yield a potent oxidant such as peroxinitrite (OONO-) which together with Mb-Fe4+ oxidizes DHR. Further increases in NO fluxes significantly inhibited DHR oxidation (80%) via the NO-dependent inhibition of Mb-Fe4+ formation. Additional studies using methemoglobin (Hb-Fe3+) catalyzed oxidative reactions yielded virtually identical results. We conclude that in the presence of a hemoprotein such as myoglobin or hemoglobin, NO may promote or inhibit oxidation reactions depending upon the relative fluxes of O-2(-), H2O2, and NO. (C) 1998 Academic Press.