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DOI: 10.1039/A606782D (Paper) Reference Section for: J. Chem. Soc., Dalton Trans., 1997, 795-802

Nuclear magnetic resonance studies of pyridine binding to cytochrome c[hair space]

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Gaohua Liu, Yu Chen and Wenxia Tang

Abstract

The binding of pyridine by displacement of the methionine-80 residue from heart ferricytochrome c (cyt c) has been studied by 1H NMR spectroscopy. Owing to the low stability of the pyridine-bound form of cytochrome c, the so-called alkaline isomer (lysine form) of cyctochrome c appears at pH > 6 in the presence of 0.45 mol dm-3 pyridine, in contrast to the native lysine form which only appears at pH > 9. A mixture of native cytochrome c and pyridine without the lysine form can only be obtained at pH < 6. The bound pyridine is replaced by a lysine as the pH value increases. Native cytochrome c, pyridine-bound cytochrome c and the lysine form can exist simultaneously at neutral pH. A pure lysine form can be obtained at pH > 8 in the presence of 1.27 mol dm-3 pyridine. Using the known resonance assignments for native cytochrome c, some hyperfine-shifted resonances arising from haem peripheral protons and two axial ligands and some side-chain resonances of the aliphatic and aromatic protons of pyridine-ligated cyt c (pcyt c) have been assigned. The experimental dipolar shifts for protons belonging to the non-co-ordinated residues have allowed the identification of the plausible orientation and magnitude of the g tensor. The sources of the asymmetric spin density distribution in the haem group of pcyt c are discussed. The substitution reaction rate constants and the equilibrium constant for pyridine binding to cyt c have been evaluated.

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