Characterization and NMR studies of a novel cytochrome c isolated from Methylophilus methylotrophus which shows a redox-linked change of spin state

Abstract

A new heme c protein has been purified from Methylophilus methylotrophus. It is a monomer of 15 kDa and contains one heme c per molecule; the isoelectric point is 8.7 and the oxidation-reduction midpoint potential is −60 mV at pH 7.6. Optical absorption spectra are characteristic of a high-spin hemoprotein in the reduced form and of a low-spin c-type cytochrome in the oxidized form. NMR shows that the iron has a histidine ligand, but there is no methionine ligand. The heme methyl resonances of the oxidized form have strongly pH-dependent shifts with a pKa of about 5.5. A significant change in the conformation of the oxidized protein is detected by NMR at alkaline pH, though optical spectroscopy is insensitive to the change. The reduced form undergoes an alkaline transition with a change of spin state which is detected both by NMR and by optical spectroscopy. The new cytochrome c binds CO in the reduced form and both forms react with CN−, but only at high pH. No heme-binding site was found in the first 44 N-terminal residues.