Structural Basis for the Molecular Properties of Cytochrome c_6

摘要

This is a thorough biochemical, spectroscopic, electrochemical, and structural study of a cytochrome c_6 isolated from the filamentous green alga Cladophora glomerata. The protein sequence, elucidating using chemical and mass spectrometric techniques, features 91 amino acids and the characteristic CXXCH heme-binding motif found in c-type cytochromes. The protein is monomeric in both oxidation forms, thereby putting in question a functional role for protein dimerization. Direct electrochemical measurements established, for the first time, the kinetic and thermodynamic data for the redox process in a cytochrome c_6. In particular, the quasi-reversible and diffusion-controlled redox process is accompanied by negative enthalpy and entropy changes, resulting in an E~o' value of 0.352 V at 298 K. The pH-dependent properties of the oxidized protein, detected by UV-visible, NMR, and direct cyclic voltammetry, indicate the presence of two acid-base equilibria occurring in the acidic (pK_a = 4.5) and alkaline regions (pK_a = 9.0). NMR and electronic spectra allowed the assignment of these equilibria to deprotonation of heme propionate-7 and to replacement of the axial methionine with anothe rligand, respectively. The 1.3 A resolution X-ray structure of the oxidized protein, revealing a fold typical for class I cytochromes, suggests that the conserved Lys60 replaces the axial methionine at pH > 9. The heme solvent accessibility is low, and no water molecules were found in the vicinity of the axial ligands of the heme Fe. A structure-based alignment of cytochromes c_6, and the direct comparison of their structures, indicate a substantial degree of identify between the tertiary structures and suggest patches involved in protein-protein interaction. In particular, the surface electrostatic potential of cytochromes c_6 features a hydrophobic region around the heme cofactor, and a backside surface rich in negative charges.