Some aspects of pH and temperature dependence of the NMR spectra of cytochrome c

Abstract

The high resolution NMR spectrum of ferricytochrome c is pH dependent. The hyperfine shifted absorptions titrate with pK's ∼9 and ∼ 11 on the alkaline side of neutrality but stay characteristic of a low spin heme iron. Below pH ∼3, all shifted absorptions are broadened beyond detection, indicating the presence of a high spin heme iron. From the relaxation rate of H2O protons below pH ∼3, one finds that the heme iron is exposed to the solvent, and has a rapidly exchanging water ligand. The studies further suggest that above pH ∼9, the iron-methionine coordination is no longer present and methionine is replaced, perhaps by a lysine. From the pH and temperature dependence of the upfield region of the NMR spectrum of reduced cytochrome c, it appears that a hydrogen bond involving a threonine or a methionine is at least partly responsible for keeping the crevice structure of the reduced protein closed. At pH ∼12, this bond is reversibly broken, though methionine still remains coordinated to the heme in the reduced state. © 1971.