title: Structural basis for the molecular properties of cytochrome c6.
abstract: 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, elucidated 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 degrees ' 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 another ligand, 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 identity 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.
authors.affiliation: Department of Agro-Environmental Science and Technology, University of Bologna, Via Filippo Re 8, Italy.
authors.forename: Alexander
authors.initials: A
authors.lastname: Dikiy
authors.forename: Wesley
authors.initials: W
authors.lastname: Carpentier
authors.forename: Isabel
authors.initials: I
authors.lastname: Vandenberghe
authors.forename: Marco
authors.initials: M
authors.lastname: Borsari
authors.forename: Niyaz
authors.initials: N
authors.lastname: Safarov
authors.forename: Elena
authors.initials: E
authors.lastname: Dikaya
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Beeumen
authors.forename: Stefano
authors.initials: S
authors.lastname: Ciurli
mesh_terms: D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry / Q000302:isolation & purification; D000460:Chlorophyta / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D003580:Cytochromes / Q000737:chemistry* / Q000302:isolation & purification; D045348:Cytochromes f; D004563:Electrochemistry; D006418:Heme / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
chemical_list: D001051:Apoenzymes; D003580:Cytochromes; D006418:Heme; D045348:Cytochromes f