MITOCHONDRIAL CREATINE-KINASE IS A PRIME TARGET OF PEROXYNITRITE-INDUCED MODIFICATION AND INACTIVATION

The reaction of peroxynitrite (PN) with sarcomeric mitochondrial creat ine kinase (Mi(b)-CK; EC 2.7.3.2) was observed at different stages of complexity (i) with purified Mi-CK, (ii) with enzyme bound on isolated mitoplasts, and (iii) within intact respiring mitochondria. Creatine- stimulated respiration was abolished by PN concentrations likely to be physiological and far before the respiratory chain itself was affecte d, thus demonstrating that Mi-CK is a prime target for inactivation by PN in intact mitochondria. The inactivation by PN of Mi CK was revers ed by 22% with 2-mercaptoethanol. More remarkable protective effects w ere noticed with the full set of CK substrates, e.g. 30 and 50% protec tion with MgATP plus creatine and MgADP plus phosphocreatine, respecti vely, but not with each substrate alone. These data indicate an involv ement of the active-site Cys-278 residue of Mi CK in this process. Fur thermore, changes in endogenous tryptophan fluorescence intensity and spectral changes after reaction of Mi-CK with PN suggest additional mo difications of Trp and Tyr residues. PN-inactivated Mi-CK can no longe r be efficiently converted into dimers by incubation with reagents ind ucing a transition state analog complex at the active site. Thus, obvi ously, upon reaction of octameric Mi-CK with PN, the octamer-dimer equ ilibrium of Mi-CK is also affected. The consequences for cellular ener gy homeostasis and calcium handling are discussed.