Structure of zinc-substituted cytochrome c: nuclear magnetic resonance and optical spectroscopic studies.

Optical and proton nuclear magnetic resonance (NMR) studies were carried out to assess the structure of the polypeptide chain and metal ligation in zinc-substituted horse heart cytochrome c (Zn Cyt c). The 1D- and 2D-NMR (COSY, TOCSY, and NOESY) spectra allowed the assignment of proton resonances in 67 amino acid residues. These residues arose from all structural elements of the protein, alpha-helices, beta-turns, and segments of the protein with no defined secondary structure. Small deviations of the chemical shifts of Zn Cyt c proton resonances from native Fe(II) Cyt c of less than 0.1 ppm are due to not fully matching solvent conditions. Differences in the chemical shifts between the two proteins in the range 0.10-0.20 ppm are not clustered and are observed not only in the vicinity of the Zn porphyrin but also on distant surface locations of the cytochrome. The resonance positions of the bridge protons, from the thioether bonds of the porphyrin with Cys 14 and Cys 17, were conserved in Zn Cyt c. Similarly, the Met 80 and His 18 protons had chemical shifts supporting the proposal that His 18 and Met 80, as for Fe(II) Cyt c, may provide the axial ligation in the Zn protein and that zinc may be in an unusual hexacoordinated geometry. Chemical shifts from proton resonances of alternative axial ligands of misfolded cytochrome like His 33, Lys 79, and Phe 82 were found to be the same as in the Fe(II) protein, excluding the possibility of their axial ligation to Zn. The His 18-Zn-Met 80 ligation was also consistent with data from absorption and luminescence studies. We conclude that Zn Cyt c is an adequate structural model for Fe(II) Cyt c as both share the same overall structure, including axial ligands, environment in the porphyrin vicinity, and the same binding interface with redox partners.