Effect of varying polyglutamate chain length on the structure and stability of ferricytochrome c

The effect of varying polyglutamate chain length on local and global stability of horse heart ferricytochrome c was studied using scanning calorimetry and spectroscopy methods. Spectral data indicate that polyglutamate chain lengths equal or greater than eight monomer units significantly change the apparent pK_a for the alkaline transition of cytochrome c. The change in pK_a is comparable to the value when cytochrome c is complexed with cytochrome bc₁. Glutamate and diglutamate do not significantly alter the temperature transition for cleavage of the Met ⁸⁰-heme iron bond of cytochrome c. At low ionic strength, polyglutamates consisting of eight or more glutamate monomers increase midpoint of the temperature transition from 57.3±0.2 to 66.9±0.2°C. On the other hand, the denaturation temperature of cytochrome c decreases from 85.2±0.2 to 68.8±0.2°C in the presence of polyglutamates with number of glutamate monomers n≳8. The rate constant for cyanide binding to the heme iron of cytochrome c of cytochrome c-polyglutamate complex also decreases by ∼42.5% with n≳8. The binding constant for the binding of octaglutamate (m.w.∼1000) to cyt c was found to be 1.15×10 ⁵ M^-1 at pH 8.0 and low ionic strength. The results indicate that the polyglutamate (n≳8) is able to increase the stability of the methionine sulfur-heme iron bond of cytochrome c in spite of structural differences that weaken the overall stability of the cyt c at neutral and slightly alkaline pH.