CHARACTERIZATION OF THE HEME ENVIRONMENT IN METHYLOPHILUS-METHYLOTROPHUS FERRICYTOCHROME-C'' BY H-1-NMR

Two-dimensional NMR techniques have been used to assign proton resonan ces in the haem cavity of Methylophilus methylotrophus cytochrome c'', a monohaem protein with bis-histidinyl ligation which has been shown to couple electron and proton transfer. All the assignments were made directly for the oxidized paramagnetic form of the cytochrome. Nearly all of the haem protons (90%) and the protons of both axial ligands ha ve been assigned; the side-chain protons from four other residues in t he haem pocket have also been identified. The data indicate a highly s ymmetric unpaired-electron distribution in the haem group, which agree s with a perpendicular orientation of the axial imidazole planes. The two haem propionate groups have contrasting degrees of exposure to the solvent, with the propionate group at position 13 being highly expose d. To obtain information on the dynamics of the haem environment, meas urements of the H-1/H-2-exchange rates of amide protons located in the haem cavity were performed. The two faces of the haem are found to di ffer markedly with respect to water accessibility. All of this informa tion, together with additional protein sequencing data, indicates that His52 remains attached upon reduction and that the redox-linked proto nation occurs via a channel running through the haem cleft on the oppo site face.