Characterization of zinc-substituted cytochrome c by circular dichroism and resonance Raman spectroscopic methods

Iron(III) in cytochrome c is replaced with zinc(II) by a modification of a method published by others, and the procedure is described in full detail. Three forms of cytochrome c-those containing iron(III), iron(II), and zinc(II)-are examined by circular dichroism spectroscopy and resonance Raman spectroscopy. Spectra of both kinds show that introduction of zinc(II) ions does not appreciably alter the overall structure and conformation of cytochrome c. Resonance Raman spectra indicate the size of the porphyrin "core" that is inconsistent with six-coordination and consistent with five-coordination. Unlike the iron(III) and iron(II) ions, which are bound to two axial ligands (His 18 and Met 80), the zinc(II) ion in cytochrome c seems to be bound to only one, most probably His 18. Evidence pertaining to the question of axial coordination is discussed.