Abstract
Proton NMR studies of Saccharomyces cerevisiae (bakers yeast) isozyme-1 monomer and dimer ferricytochrome c have been carried out. The dimer is formed via a disulfide bridge between the Cys-102 residues of monomer proteins. Nuclear Overhauser effect (NOE) experiments have led to resonance assignments for many of the heme and axial ligand (Met-80; His-18) protons in both protein forms. Resonances of the following amino acids have also been assigned in both forms: Phe-10; Pro-30; Phe-82; Trp-59; Leu-68. The proton NOE connectivity patterns of the monomer of yeast isozyme-1 ferricytochrome c are similar to those of horse, tuna, and yeast isozyme-2 ferricytochromes c, even though the observed hyperfine resonance spectra are significantly different for the various cytochromes. The pattern of dimer proton hyperfine resonances is distinct from the isozyme-1 monomer pattern, which indicates that the formation of a disulfide bridge via Cys-102 is detected at the heme site, approximately 10 A distant. It appears that a specific structural change is induced upon dimerization, which, in turn, causes specific perturbations in the vicinity of the heme. However, the general features of the NOE connectivity pattern in the dimer are the same as for the monomer indicating that dimerization does not result in drastic structural disruption. Furthermore, the 1H NMR spectrum of the dimer can be mimicked by the monomer form that results when the -SH group of Cys-102 is chemically modified with certain types of bulky, or hydrophilic reagents (i.e. 5,5'-dithiobis[2-nitrobenzoate], indicating that perturbations of the yeast isozyme-1 ferricytochrome c proton resonance spectrum observed upon dimerization are essentially due to changes in intramolecular, rather than intermolecular, interactions. These results suggest that a possible regulatory site for yeast isozyme-1 cytochrome c exists at position 102, which could conceivably have a physiological role in altering the conformation of the molecule.
Highlights
Proton NMR studies of Saccharomyces cerevisiae isozyme-2 [1, 2]
The primary sequence homology between iso-1 and iso-2 isrelatively high [4,17,18], but iso-1 is unique in thatit possesses a single cysteine at the tra are significantly different for the various cytoposition corresponding to 102 in the primary sequence of tuna chromes
These results suggest that paossible reg- Resonance assignments have been madeusing the one-dimenulatory site foyreast isozyme-1 cytochrome c exists at sional nuclear Overhauser effect (NOE)‘ and they are composition 102, which could conceivably have a physiological role in altering the conformation of the molecule
Summary
For the following presentation of proton hyperfine resonance assignments the heme c structure, complete with our labeling scheme, is presented Finig. lA.The iso-1cytochrome c crystal structure has just recently been published [29] but its structural coordinates file is currently unavailable from the National Protein Data Bank. In spectra such as these tihnedividual shifts of each species are unaffected by the relative amounts of monomer and dimer present, indicating that the monomer and dimer undergo no further noncovalent interactions under the conditions employed. Nearallyl of the resolved hyperfine resonances appear tobe single resonances at 361 MHz, splitting of theheme3-CH3issometimes observed, at higherfields (Fig. 3) This indicates that protons of corresponding heme substituentsin eachmonomer unit of the dimerexhibitessentiallyidentical magnetic environments, within the resolution of our spectrometer,which means that, in thiscase, structural differences in thevicinity of the heme, between the two monomer units within the intacdt imer, are minor. It is interesting to note that theoverall resonance pattern of the iso-1 dimer more closely resembles the Proton NMR Study of Yeast Iso-1 Monomer and Disulfide Dimer
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