Molten-globule and other conformational forms of zinc cytochrome C. Effect of partial and complete unfolding of the protein on its electron-transfer reactivity.

To test the effect of protein conformation on reactivity, we use laser flash photolysis to compare the electron-transfer properties of the triplet state of zinc-substituted cytochrome c, designated (3)Zncyt, in the folded forms at low (F(low)) and high (F(high)) ionic strength, molten-globule (MG) form, and the forms unfolded by acid (U(acid)) and urea (U(urea)) toward the following four oxidative quenchers: Fe(CN)(6)(3-), Co(acac)(3), Co(phen)(3)(3+), and iron(III) cytochrome c. We characterize the conformational forms of Zncyt on the basis of the far-UV circular dichroism, Soret absorption, and rate constant for natural decay of the triplet state. This rate constant in the absence of quencher increases in the order F(high) < F(low) < MG < U(acid) < U(urea) because the exposure of porphyrin to solvent increases as Zncyt unfolds. Bimolecular rate constants for the reaction of (3)Zncyt with the four quenchers show significant effects on reactivity of electrostatic interactions and porphyrin exposure to solvent. This rate constant at the ionic strength of 20 mM increases upon unfolding by urea and acid, respectively, as follows: 1340-fold and 466-fold when the quencher is Co(phen)(3)(3+) and 168-fold and 36-fold when the quencher is cyt(III). To compare reactivity of (3)Zncyt in the F(low), F(high), MG, U(acid), and U(urea) forms without complicating effects of electrostatic interactions, we used the electroneutral quencher Co(acac)(3). Indeed, reactivity of folded (3)Zncyt with Co(acac)(3) was independent of ionic strength. Reactivity of (3)Zncyt with Co(acac)(3) upon partial and complete unfolding increases 10-fold, 54-fold, and 64-fold in the molten-globule, urea-unfolded, and acid-unfolded forms.