Polyanion binding to cytochrome c probed by resonance Raman spectroscopy.
The interaction of ferricytochrome c with negatively charged heteropolytungstates was studied by resonance Raman spectroscopy. In analogy to previous findings on ferricytochrome c bound to other types of charged interface (Hildebrandt, P. and Stockburger, M. (1989) Biochemistry 28, 6710-6721, 6722-6728), it was shown that in these complexes the conformational states I and II are stabilized. While in state I, the structure is the same as is in the uncomplexed heme protein, in state II three different coordination configurations coexist, i.e., a six-coordinated low-spin, a five-coordinated high-spin and a six-coordinated high-spin form. These configurations constitute thermal coordination equilibria whose thermodynamic properties were determined. The detailed analysis of the low-frequency resonance Raman spectra reveals that in state II the heme pocket assumes an open structure leading to a significantly higher flexibility of the heme group compared to the native ferricytochrome c. It is concluded that these structural changes are the result of Coulombic attractions between the polyanions and the lysine residues around the exposed heme edge which destabilize the heme crevice. Modifications of these interactions upon variation of the ionic strength, the pH or the type of the polytungstate are sensitively reflected by changes of the coordination equilibria in state II as well as of the conformational equilibrium of state I and state II. The conformational changes in state II significantly differ from those associated with the alkaline transition of ferricytochrome c. However, there are some structural similarities between the acid form of the heme protein stable below pH 2.5 in aqueous solution and the six-coordinated high-spin configuration of the bound ferricytochrome c at neutral pH (state II). This suggests that electrostatic interactions with the heteropolytungstates perturb the ionic equilibria of those amino acid side chains which are involved in the acid-induced transition leading to a significant upshift of the apparent pKa.
Hildebrandt
P
P
Max-Planck-Institut für biophysikalische Chemie, Abteilung Spektroskopie, Göttingen, F.R.G.
D000818:Animals; D000838:Anions; D003574:Cytochrome c Group / Q000378:metabolism*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D009206:Myocardium / Q000378:metabolism; D009942:Organometallic Compounds / Q000378:metabolism* / Q000494:pharmacology; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman / Q000379:methods; D014414:Tungsten / Q000378:metabolism* / Q000494:pharmacology
D000838:Anions; D003574:Cytochrome c Group; D009942:Organometallic Compounds; D014414:Tungsten