A CHEMICAL MODIFICATION OF CYTOCHROME-C LYSINES LEADING TO CHANGES INHEME IRON LIGATION

Although 13 lysines of horse cytochrome c are invariant, and three mor e are extremely conserved, the modification of their side-chain epsilo n-amino groups by beta-thiopropionylation caused important changes in protein properties for only three of them; lysines 72,73 and 79. Optic al spectroscopy, electron and nuclear paramagnetic resonance, electron spin echo envelope modulation, and molecular weight studies, as well as the unique features of their reaction with cytochrome-c oxidase, in dicate that in the oxidized state the modification of these lysines re sulted in equilibria between two different states of iron ligation: th e native state, in which the metal is coordinated by the methionine-80 sulfur, and a new state in which this ligand is displaced by the sulf hydryl groups of the elongated side chains. The reduction potentials o f the TP Lys-72 and the TP Lys-79 derivatives were 201 and 196 millivo lt, respectively, indicating that the equilibria favored the sulfhydry l ligated state by 1.5 and 1.7 kcal/mol, respectively. In the ferric s tate, the protein modified at lysine 72 remained stable as a monomer, but that modified at lysine 73 dimerized rapidly through disulfide bon d formation, while the TP Lys-79 cytochrome c dimerized with a half-ti me of approx. 3 h, both recovering the native-like iron ligation. By c ontrast, in the ferrous state the monomeric state and the native ligat ion were preserved in all cases, indicating that the affinity of the c ytochrome-c ferrous iron for the methionine-80 sulfur is particularly strong. The dimerized derivatives lost most, but not all, of the capab ility of the native protein for electron transfer from ascorbate-TMPD to cytochrome-c oxidase.