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 Thiobacillus versutus is replaced by another residue at high pH. This transition is similar to the alkaline transition in mitochondrial cytochrome c. To investigate the possible role of lysine 99 in this process, this residue has been mutated to a glutamate. The mutation causes the apparent pKa of the transition to decrease from 11.2 in wild type to 10.8 in Lys99Glu cytochrome c-550. This destabilization of the native form is ascribed to the absence of the hydrogen bond between the epsilon-amine group of Lys99 and the carbonyl of Lys54 in the mutant protein. The 1H-NMR spectrum of Lys99Glu ferricytochrome c-550 at alkaline pH still shows resonance positions of the heme methyl peaks that are characteristic of the alkaline form. These results strongly suggest that Lys99 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 that in the latter protein the homologous Lys79 can act as a ligand in the alkaline form [1993, J. Am. Chem. Soc. 115, 7507-7508]. In the EPR spectrum of Lys99Glu cytochrome c-550 the species with Met-His coordination (gz = 3.27) is replaced by two forms with gz = 3.45 and 3.20 in the alkaline form (pH > or = 10.6). At pH > 11 yet another form is observed with g-values 2.87, 2.18 and 1.60, tentatively identified as a species with a lysine-histidinate coordination of the heme iron.
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Gorlaeus Laboratories, Leiden Institute of Chemistry, The Netherlands.
Warmerdam
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Teixeira
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D003574:Cytochrome c Group / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008239:Lysine / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D013855:Thiobacillus / Q000201:enzymology*
D003574:Cytochrome c Group; D008024:Ligands; C031732:cytochrome C-550; D008239:Lysine