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 met hionine and a histidine as axial ligands. In order to study the charac teristics of a possible lysine-histidine ligation in a heme protein, t he methionine has been replaced by a lysine. This residue acts as a li gand between pH 3 and 12. The midpoint potential of the mutant has shi fted -329 mV compared to wild type, but apart from this shift the pH d ependence of the midpoint potential is unchanged, suggesting that the large drop is caused by specific ligand effects and not by protein ref olding. While the EPR spectrum of wild-type cytochrome c-550 shows one species with g(z) = 3.35, in the spectrum of the mutant two species o ccur with g(z) values of 3.53 and 3.30. The intensity ratio of both sp ecies depends on the presence of organic cosolvents. In the low freque ncy region (-4 to -1 ppm) of the H-1 NMR spectrum of mutant ferrocytoc hrome c-550, four one-proton peaks replace the resonances of the ligan d methionine side chain protons. Using two-dimensional NMR spectroscop y (COSY and NOESY), these protons and five others have been assigned t o the lysine ligand. The spectroscopic results obtained for this mutan t show similarities with those observed for the alkaline form of cytoc hrome c, supporting the Lys-His ligation proposed for this protein. Th e data are consistent with the evidence for amine ligation in cytochro me f: the EPR spectrum of M100K cytc-550 is similar to that of cytochr ome f. However, the NMR spectra show significant differences. At high pH wild-type cytochrome c-550 shows a complex EPR behavior: at pH >10 new species are observed, with g(z) 3.45 (possibly due to a lysine-his tidine ligation) and g(z) approximate to 3.2. At pH >11 a species with g(z) = 2.93, g(y) = 2.23, and g(x) = 1.67 is observed. This new form is also seen for the M100K mutant and may represent lysine-histidinate ligation of the heme iron.