The heme iron coordination of unfolded ferric and ferrous cytochrome c in neutral and acidic urea solutions. Spectroscopic and electrochemical studies

Abstract

The heme iron coordination of unfolded ferric and ferrous cytochrome c in the presence of 7–9 M urea at different pH values has been probed by several spectroscopic techniques including magnetic and natural circular dichroism (CD), electrochemistry, UV–visible (UV–vis) absorption and resonance Raman (RR). In 7–9 M urea at neutral pH, ferric cytochrome c is found to be predominantly a low spin bis–His-ligated heme center. In acidic 9 M urea solutions the UV–vis and near-infrared (NIR) magnetic circular dichroism (MCD) measurements have for the first time revealed the formation of a high spin His/H 2O complex. The p K a for the neutral to acidic conversion is 5.2. In 9 M urea, ferrous cytochrome c is shown to retain its native ligation structure at pH 7. Formation of a five-coordinate high spin complex in equilibrium with the native form of ferrous cytochrome c takes place below the p K a 4.8. The formal redox potential of the His/H 2O complex of cytochrome c in 9 M urea at pH 3 was estimated to be −0.13 V, ca. 100 mV more positive than E°′ estimated for the bis–His complex of cytochrome c in urea solution at pH 7.