THE ROLE OF LYSINE-99 OF THIOBACILLUS-VERSUTUS CYTOCHROME C-550 IN THE ALKALINE TRANSITION

The methionine ligand of the heme iron in ferricytochrome c-550 from T hiobacillus versutus is replaced by another residue at high pH. This t ransition is similar to the alkaline transition in mitochondrial cytoc hrome c. To investigate the possible role of lysine 99 in this process , this residue has been mutated to a glutamate. The mutation causes th e apparent pK(a) of the transition to decrease from 11.2 in wild type to 10.8 in Lys(99)Glu cytochrome c-550. This destabilization of the na tive form is ascribed to the absence of the hydrogen bond between the E-amine group of Lys(99) and the carbonyl of Lys(54) in the mutant pro tein. The H-1-NMR spectrum of Lys(99)Glu ferricytochrome c-550 at alka line pH still shows resonance positions of the heme methyl peaks that are characteristic of the alkaline form. These results strongly sugges t that Lys(99) does not act as a ligand in the high-pH form, contrary to the case of yeast iso-1-cytochrome c. Evidence has been presented t hat in the latter protein the homologous Lys(79) can act as a ligand i n the alkaline form [1993, J. Am. Chem. Sec. 115, 7507-7508]. In the E PR spectrum of Lys(99)Glu cytochrome c-550 the species with Met-His co ordination (g(z) = 3.27) is replaced by two forms with g(z) = 3.45 and 3.20 in the alkaline form (pH greater than or equal to 10.6). At pH > 11 yet another form is observed with g-values 2.87, 2.18 and 1.60, te ntatively identified as a species with a lysine-histidinate coordinati on of the heme iron.