NMR study of the alkaline isomerization of ferricytochrome c

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Abstract
The pH‐induced isomerization of horse heart cytochrome c has been studied by 1H NMR. We find that the transition occurring in D2O with a pK a measured as 9.5±0.1 is from the native species to a mixture of two basic forms which have very similar NMR spectra. The heme methyl peaks of these two forms have been assigned by 2D exchange NMR. The forward rate constant (native to alkaline cytochrome c) has a value of 4.0±0.6 s−1 at 27°C and is independent of pH; the reverse rate constant is pH‐dependent. The activation parameters are ΔH ‡ = 12.8±0.8 kcal·mol1, ΔS ‡ = − 12.9±2.0 e.u. for the forward reaction and ΔH ‡ = 6.0±0.3 kcal·mol−1, ΔS ‡ = − 35.1±1.3 e.u. for the reverse reaction (pH*=9.28). ΔH° and ΔS° for the isomerization are 6.7±0.6 kcal·mol−1 and 21.9±1.0 e.u., respectively.