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Insights into the alkaline transformation of ferricytochrome c from ¹H NMR studies in 30% acetonitrile–water

Department of Chemistry, Northeastern University, Boston, Massachusetts 02115, USA

Reprint requests to: Patricia Ann Mabrouk, Department of Chemistry, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA; e-mail: p.mabrouk{at}neu.edu; fax: (617) 373-8795.

Recently, we found that ferricytochrome c (ferricyt c) undergoes significant structural changes in mixed aqueous–nonaqueous media, resulting in the formation of a mixture of alkaline-like species. The equilibrium composition of this mixture of species is dependent on the dielectric constant of the mixed solvent medium. One-dimensional (1D) and two-dimensional (2D) ¹H nuclear magnetic resonance (NMR) methods have now been used to study these alkaline-like forms in 30% acetonitrile–water solution. A native-like (M80-ligated) III* form, two lysine-ligated forms (IVa* and IVb*), and a hydroxide-ligated form (V*) were observed. Heme proton resonance assignments for these forms were accomplished using 1D ¹H NMR and 2D nuclear Overhauser effect spectroscopy methods at 20°C and 35°C. The chemical exchange between the alkaline forms in 30% acetonitrile solution facilitated heme proton resonance assignments. Based on examination of the heme proton chemical shifts and several highly conserved amino acid residues, the electronic structure, secondary structure, and hydrogen bond network in the vicinity of the heme in the III* form were found to be intact. Similarly, the heme electronic structure of the IVa* form was found to be comparable to that of the IVa form. Differences in the order of the heme methyl resonances in the IVb* form, however, suggest that the heme active site in this form is somewhat different from that observed in aqueous alkaline solution. In addition, resonance assignments for the 8- and 3-methyl heme protons were made for the hydroxide-ligated V* form for the first time. The observation of chemical exchange peaks between all species except IVb* and IVa* or V* was used to propose an exchange pathway between the different forms of ferricyt c in 30% acetonitrile solution. This pathway may be biologically significant because ferricyt c, which resides in the intermembrane space of mitochondria, is exposed to medium of relatively low dielectric constant when it interacts with the mitochondrial membrane.

Keywords: Cytochrome c; alkaline transition; NMR; nonaqueous enzymology; heme protein

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