Characterization of mutant Met100Lys of cytochrome c-550 from Thiobacillus versutus with lysine-histidine heme ligation.
The heme iron in cytochrome c-550 from Thiobacillus versutus has a methionine and a histidine as axial ligands. In order to study the characteristics of a possible lysine-histidine ligation in a heme protein, the methionine has been replaced by a lysine. This residue acts as a ligand between pH 3 and 12. The midpoint potential of the mutant has shifted -329 mV compared to wild type, but apart from this shift the pH dependence of the midpoint potential is unchanged, suggesting that the large drop is caused by specific ligand effects and not by protein refolding. While the EPR spectrum of wild-type cytochrome c-550 shows one species with gz = 3.35, in the spectrum of the mutant two species occur with gz values of 3.53 and 3.30. The intensity ratio of both species depends on the presence of organic cosolvents. In the low frequency region (-4 to -1 ppm) of the 1H NMR spectrum of mutant ferrocytochrome c-550, four one-proton peaks replace the resonances of the ligand methionine side chain protons. Using two-dimensional NMR spectroscopy (COSY and NOESY), these protons and five others have been assigned to the lysine ligand. The spectroscopic results obtained for this mutant show similarities with those observed for the alkaline form of cytochrome c, supporting the Lys-His ligation proposed for this protein. The data are consistent with the evidence for amine ligation in cytochrome f: the EPR spectrum of M100K cytc-550 is similar to that of cytochrome f. However, the NMR spectra show significant differences.(ABSTRACT TRUNCATED AT 250 WORDS)
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Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden University, The Netherlands.
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D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013855:Thiobacillus / Q000737:chemistry*
D003574:Cytochrome c Group; D006418:Heme; D006639:Histidine; C031732:cytochrome C-550; D008715:Methionine; D008239:Lysine