title: Structure-function relationships in human cytochrome c: The role of tyrosine 67.
abstract: Spectroscopic and functional properties of human cytochrome c and its Tyr67 residue mutants (i.e., Tyr67His and Tyr67Arg) have been investigated. In the case of the Tyr67His mutant, we have observed only a very limited structural alteration of the heme pocket and of the Ω-loop involving, among others, the residue Met80 and its bond with the heme iron. Conversely, in the Tyr67Arg mutant the Fe-Met80 bond is cleaved; consequently, a much more extensive structural alteration of the Ω-loop can be envisaged. The structural, and thus the functional modifications, of the Tyr67Arg mutant are present in both the ferric [Fe(III)] and the ferrous [Fe(II)] forms, indicating that the structural changes are independent of the heme iron oxidation state, depending instead on the type of substituting residue. Furthermore, a significant peroxidase activity is evident for the Tyr67Arg mutant, highlighting the role of Arg as a basic, positively charged residue at pH7.0, located in the heme distal pocket, which may act as an acid to cleave the O-O bond in H2O2. As a whole, our results indicate that a delicate equilibrium is associated with the spatial arrangement of the Ω-loop. Clearly, Arg, but not His, is able to stabilize and polarize the negative charge on the Fe(III)-OOH complex during the formation of Compound I, with important consequences on cytochrome peroxidation activity and its role in the apoptotic process, which is somewhat different in yeast and mammals.
authors.affiliation: Department of Chemistry "Ugo Schiff", University of Florence, Sesto Fiorentino, FI, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Tognaccini
authors.affiliation: Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Rome, Italy; Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems (CIRCMSB), Bari, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Ciaccio
authors.affiliation: Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: D'Oria
authors.affiliation: Department of Sciences, University of Rome Tre, Rome, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Cervelli
authors.affiliation: Department of Chemistry "Ugo Schiff", University of Florence, Sesto Fiorentino, FI, Italy.
authors.forename: Barry D
authors.initials: BD
authors.lastname: Howes
authors.affiliation: Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Rome, Italy; Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems (CIRCMSB), Bari, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Coletta
authors.affiliation: Department of Sciences, University of Rome Tre, Rome, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Mariottini
authors.affiliation: Department of Chemistry "Ugo Schiff", University of Florence, Sesto Fiorentino, FI, Italy; Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems (CIRCMSB), Bari, Italy. Electronic address: giulietta.smulevich@unifi.it.
authors.forename: Giulietta
authors.initials: G
authors.lastname: Smulevich
authors.affiliation: Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Rome, Italy. Electronic address: fiorucci@uniroma2.it.
authors.forename: Laura
authors.initials: L
authors.lastname: Fiorucci
mesh_terms: D002942:Circular Dichroism; D045304:Cytochromes c / Q000737:chemistry* / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding; D013059:Spectrum Analysis, Raman; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000502:physiology*
chemical_list: D008024:Ligands; D014443:Tyrosine; D045304:Cytochromes c
keywords: Apoptosis; Circular dichroism; Ligand binding kinetics; Peroxidase activity; Resonance Raman; Site directed mutagenesis