Mitochondrial creatine kinase is a prime target of peroxynitrite-induced modification and inactivation.
The reaction of peroxynitrite (PN) with sarcomeric mitochondrial creatine kinase (Mib-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 affected, thus demonstrating that Mi-CK is a prime target for inactivation by PN in intact mitochondria. The inactivation by PN of Mi-CK was reversed by 22% with 2-mercaptoethanol. More remarkable protective effects were noticed with the full set of CK substrates, e.g. 30 and 50% protection with MgATP plus creatine and MgADP plus phosphocreatine, respectively, but not with each substrate alone. These data indicate an involvement of the active-site Cys-278 residue of Mi-CK in this process. Furthermore, changes in endogenous tryptophan fluorescence intensity and spectral changes after reaction of Mi-CK with PN suggest additional modifications of Trp and Tyr residues. PN-inactivated Mi-CK can no longer be efficiently converted into dimers by incubation with reagents inducing a transition state analog complex at the active site. Thus, obviously, upon reaction of octameric Mi-CK with PN, the octamer-dimer equilibrium of Mi-CK is also affected. The consequences for cellular energy homeostasis and calcium handling are discussed.
Stachowiak
O
O
Institute of Cell Biology, Swiss Federal Institute of Technology, ETH Zürich, CH-8093 Zürich, Switzerland.
Dolder
M
M
Wallimann
T
T
Richter
C
C
D000818:Animals; D002645:Chickens; D003402:Creatine Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D007527:Isoenzymes; D008931:Mitochondria, Muscle / Q000201:enzymology* / Q000378:metabolism; D009566:Nitrates / Q000737:chemistry*; D010085:Oxidative Phosphorylation; D013379:Substrate Specificity
D007527:Isoenzymes; D009566:Nitrates; C039642:peroxynitric acid; D003402:Creatine Kinase