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 ferricytochromes c. Under the conditions employed in this work, the stoichiometry of the predominant complex formed in solution (which totaled greater than 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 ferricytochromes c) are distinctly different from the noncovalent complexes formed between CcP and ferricytochromes c from horse and tuna. Parallel chemical cross-linking studies carried out using mixtures of cytochrome c peroxidase with horse ferricytochrome c, and cytochrome c peroxidase with yeast isozyme-1 ferricytochrome c 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.
Moench
S J
SJ
Department of Chemistry, Washington State University, Pullman 99164-4630.
Chroni
S
S
Lou
B S
BS
Erman
J E
JE
Satterlee
J D
JD
D000818:Animals; D003432:Cross-Linking Reagents; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000378:metabolism*; D006736:Horses; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014413:Tuna
D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D007527:Isoenzymes; D011522:Protons; D003578:Cytochrome-c Peroxidase