Proton NMR Comparison of Noncovalent and Covalently Cross-Linked Complexes of Cytochrome c Peroxidase with Horse, Tuna, and Yeast Ferricytochromes c

Proton NMR spectroscopy at 500 and 361 MHz has been used to characterize the noncovalent or electrostatic complexes of yeast cytochrome c peroxidase (CcP) with horse, tuna, yeast isozyme-1, and yeast isozyme-2 ferricytochromes c and the covalently cross-linked complexes of cytochrome c peroxidase with horse and yeast isozyme-1 ferricytochromesc. Under the conditions employed in this work, the stoichiometry of the predominant complex formed in solution (which totaled >90%; of complex formed) was found to be 1:1 in all cases. These studies have elucidated significant differences in the proton NMR absorption spectra and the one-dimensional nuclear Overhauser effect difference spectra of the complexes, depending on the specific species of ferricytochrome c incorporated. In particular, the results indicate that the noncovalent complexes formed between CcP and physiological redox partners (yeast isozyme-1 or yeast isozyme-2 ferricytochromesc) are distinctly different from the noncovalent complexes formed between CcP and ferricytochromesc from horse and tuna. Parallel chemical cross-linking studies carried out using mixtures of cytochromec peroxidase with horse ferricytochromec, and cytochromec peroxidase with yeast isozyme-1 ferricytochromec further emphasize such cytochrome c-dependent differences, with only the covalently cross-linked complex of physiological redox partners (cytochrome c peroxidase/yeast isozyme-1) displaying NMR spectra characteristic of a heterogeneous mixture of different 1:1 complexes. Finally, one-dimensional nuclear Overhauser effect experiments have proven valuable in selectively and efficiently probing the protein-protein interface in these complexes, including the environment around the cytochrome c heme 3-methyl group and Phe-82.