Proton magnetic resonance studies of cytochrome c peroxidase: pD dependence of the isotropically shifted resonances

Proton nuclear magnetic resonance studies of the isotropically shifted resonances of native cytochrome c peroxidase have been carried out at 360 MHz. Proton resonances extend to 84 ppm downfield and 12 ppm upfleld from 2,2-dimethyl-2-silapentane-5-sulfonate and are characteristic of high-spin iron +3 heme proteins. Between pH 8 and 4.1 the isotropic resonances exhibit dramatic pH-dependent behavior which demonstrates the presence of two acid-base interconversions. One process, with a pK_a of 5.8, is slow on the NMR time scale and probably represents a protein conformation change. This process correlates with an apparent pK_a observed in the kinetic properties of the enzymes, with the alkaline form being the enzymatically active species. A second ionization with a pK of 4.9 is fast on the NMR time scale and probably represents a true ionization.