STRUCTURE OF ZINC-SUBSTITUTED CYTOCHROME-C - NUCLEAR-MAGNETIC-RESONANCE AND OPTICAL SPECTROSCOPIC STUDIES

Optical and proton nuclear magnetic resonance (NMR) studies were carri ed out to assess the structure of the polypeptide chain and metal liga tion 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, an d segments of the protein with no defined secondary structure. Small d eviations of the chemical shifts of Zn Cyt c proton resonances from na tive Fe(II) Cyt c of less than 0.1 ppm are due to not fully matching s olvent 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 surf ace locations of the cytochrome. The resonance positions of the bridge protons, from the thioether bonds of the porphyrin with Cys 14 and Cy s 17, were conserved in Zn Cyt c. Similarly, the Met 80 and His 18 pro tons had chemical shifts supporting the proposal that His 18 and Met 8 0, as for Fe(II) Cyt c, may provide the axial ligation in the Zn prote in and that zinc may be in an unusual hexacoordinated geometry. Chemic al shifts from proton resonances of alternative axial ligands of misfo lded cytochrome Like His 33, Lys 79, and Phe 82 were found to be the s ame as in the Fe(II) protein, excluding the possibility of their axial ligation to Zn. The His Is-Zn-Met 80 ligation was also consistent wit h data from absorption and luminescence studies. We conclude that Zn C yt c is an adequate structural model for Fe(II) Cyt c as both share th e same overall structure, including axial ligands, environment in the porphyrin vicinity, and the same binding interface with redox partners .