Studies on the reaction of imidazole with cytochrome c3 from Desulfovibrio vulgaris

1. Cytochrome c₃, a unique hemoprotein with a negative redox potential and four heme groups bound to a single polypeptide chain, reacts with imidazole in the reduced state to form a low-spin ferro · imidazole complex which is spectrally characterized by a 3.1 nm blue shift in the α-peak (from 550.5 to 547.4 nm). The spectral imidazole · cytochrome c₃ complex is detectable at 77 but not at 298 K.2. Mammalian ferrocytochrome c did not undergo a spectral interaction with imidazole at either 77 or 298 K, indicating that the imidazole · cytochrome c₃ complex reflects a unique event for cytochrome c₃.3. Formation of the imidazole · cytochrome c₃ complex is strongly dependent on imidazole concentration (apparent K_d of approx. 50 mM), and is abolished in the presence of 100 mM phosphate. This latter effect is attributable to formation of an imidazole · phosphate complex. A pH titration of the imidazole · cytochrome c₃ spectral complex implicates ionization of an imidazole function (pK = 8.5).4. EPR studies at 8.5 K of ferricytochrome c₃ before and after one reduction-oxidation cycle indicate that at least two of the hemes undergo reaction with imidazole forming two different low-spin ferric heme · imidazole complexes, with significant shifts in the g values of two heme signals.5. The spectral and EPR results are consistent with formation as the primary event of a low-spin ferrocytochrome c₃ · imidazole complex in which increased hydrophobicity and protonation-deprotonation effects are contributary to the consequent lability of cytochrome c₃.