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

Recently, we found that ferricytochrome c (ferricyt c) undergoes significan t structural changes in mixed aqueous-nonaqueous media, resulting in the fo rmation of a mixture of alkaline-like species. The equilibrium composition of this mixture of species is dependent on the dielectric constant of the m ixed solvent medium. One-dimensional (1 D) and two-dimensional (2D) H-1 nuc lear magnetic resonance (NMR) methods have now been used to study these alk aline-like forms in 30% acetonitrile-water solution. A native-like (M80-lig ated) III* form, two lysine-ligated forms (IVa* and lVb*), and a hydroxide- ligated form (V*) were observed. Heme proton resonance assignments for thes e forms were accomplished using 1D H-1 NMR and 2D nuclear Overhauser effect spectroscopy methods at 20 degreesC and 35 degreesC. The chemical exchange between the alkaline forms in 30% acetonitrile solution facilitated heme p roton resonance assignments. Based on examination of the heme proton chemic al shifts and several highly conserved amino acid residues, the electronic structure, secondary structure, and hydrogen bond network in the vicinity o f the heme in the III* form were found to be intact. Similarly, the heme el ectronic structure of the IVa* form was found to be comparable to that of t he 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 somew hat different from that observed in aqueous alkaline solution. In addition, resonance assignments for the 8- and 3-methyl heme protons were made for t he hydroxide-ligated V* form for the first time. The observation of chemica l exchange peaks between all species except lVb* and IVa* or V* was used to propose an exchange pathway between the different forms of ferricyt c in 3 0% acetonitrile solution. This pathway may be biologically significant beca use ferricyt c. which resides in the intermembrane space of mitochondria, i s exposed to medium of relatively low dielectric constant when it interacts with the mitochondrial membrane.