Protonation of porphyrin in iron-free cytochrome c: Spectral properties ofmonocation free base porphyrin, a charge analogue of ferric heme

Charged groups reside mainly on protein surfaces, but for proteins that inc orporate redox centers, a charge typically exists at the prosthetic group w ithin the interior. How a protein accommodates a buried charge and the effe ct of redox changes on protein stability are thermodynamically related prob lems. To examine these problems in cytochrome c, the metal-free protein was used as a model. When pH is lowered, the neutral, monocation, and dication forms of the porphyrin are progressively formed as indicated by their char acteristic absorption spectra. Infrared studies of the protein over this pH range show that the protein remains in a predominately alpha-helical struc ture, although the carboxyl groups of the dicarboxylic amino acids become p rotonated at lower pH. The monocation porphyrin form (which has not been pr eviously reported in a protein and is a charge analogue of ferric heme) has a fluorescence maximum at 609 nm. The pKs for the respective one and two p rotonation of the porphyrin pyrrole Ns are 3.2 and 1.6 for the folded prote in, and 4.4 and 3.1 for the unfolded protein. These values indicate that th e protection of the polypeptide chain for protonation is similar to 3 kcal. (C) 1999 John Wiley & Sons, Inc.