REACTIONS OF NITRIC-OXIDE WITH MITOCHONDRIAL CYTOCHROME-C - A NOVEL MECHANISM FOR THE FORMATION OF NITROXYL ANION AND PEROXYNITRITE

The aerobic reactions of nitric oxide with cytochrome c were analysed. Nitric oxide (NO) reacts with ferrocytochrome c at a rate of 200 M-1 s(-1) to form ferricytochrome c and nitroxyl anion (NO-). Ferricytochr ome c was detected by optical spectroscopy; NO- was detected by trappi ng with metmyoglobin (Mb(3+)) to form the EPR-detectable Mb-nitrosyl c omplex, and by the formation of dimers in yeast ferrocytochrome c via cross-linking of the free cysteine residue. The NO- formed subsequentl y reacted with oxygen to form peroxynitrite, as measured by the oxidat ion of dihydrorhodamine 123. NO binds to ferricytochrome c to form the ferricytochrome c-NO complex. The on-rate for this reaction is 1.3 +/ - 0.4 x 10(3) M-1.s(-1), and the off-rate is 0.087 +/- 0.054 s(-1). Th e dissociation constant (K-d) of the complex is 22 +/- 7 mu M. These r eactions of NO with cytochrome c are likely to be relevant to mitochon drial metabolism of NO. Ferricytochrome c can act as a reversible sink for excess NO in the mitochondria. The reduction of NO to NO- by ferr ocytochrome c may play a role in the irreversible inhibition of mitoch ondrial oxygen consumption by peroxynitrite. It is generally assumed t hat peroxynitrite would be formed in mitochondria via the reaction of NO with superoxide. The finding that NO- is formed from the reaction o f NO and ferrocytochrome c provides a means of producing peroxynitrite in the absence of superoxide, via the reaction of NO- with oxygen.