Ascorbate-catalyzed reductive nitrosylation of ferric myoglobin.

Myoglobin plays a key role in oxygen storage and delivery through oxygen binding and release from the ferrous (Fe+2) heme. However, when the heme is oxidized to its ferric (Fe+3) form it can no longer bind oxygen and further oxidation leads to lipid peroxidation which can lead to a variety of pathological consequences. Ferric heme can be reduced to the ferrous form by nitric oxide (NO•) through classical reductive nitrosylation but this reaction, where a second NO• molecule binds to the reduced heme to form iron nitrosyl myoglobin, is very slow and inefficient. We have recently demonstrated that glutathione (and other thiols) can catalyze reductive nitrosylation of free ferric heme (hemin) when solubilized in albumin or red blood cell ghosts and that the resultant NO-ferroheme has potent NO• signaling properties. In this work we show this catalysis is also viable in myoglobin and that ascorbate is a better catalyst than glutathione with reductive nitrosylation occurring 1000 times faster in the presence of ascorbate compared to its absence under some conditions. Our data support a mechanism involving ascorbate-mediated heme reduction forming an ascorbyl radical. Only one NO• molecule and 0.5 ascorbate molecules are needed to convert one ferric myoglobin to one nitrosyl myoglobin molecule. Addition of NO• and ascorbate are shown to be more protective against lipid peroxidation due to myoglobin oxidation by hydrogen peroxide than either ascorbate or NO• alone. This reaction has potential application in meat preservation and treatment of ischemic reperfusion injury, various myopathies, and rhabdomyolysis.