The Met80Ala and Tyr67His/Met80Ala mutants of human cytochrome c shed light on the reciprocal role of Met80 and Tyr67 in regulating ligand access into the heme pocket.
The spectroscopic and functional properties of the single Met80Ala and double Tyr67His/Met80Ala mutants of human cytochrome c have been investigated in their ferric and ferrous forms, and in the presence of different ligands, in order to clarify the reciprocal effect of these two residues in regulating the access of exogenous molecules into the heme pocket. In the ferric state, both mutants display an aquo high spin and a low spin species. The latter corresponds to an OH-ligand in Met80Ala but to a His in the double mutant. The existence of these two species is also reflected in the functional behavior of the mutants. The observation that (i) a significant peroxidase activity is present in the Met80Ala mutants, (ii) the substitution of the Tyr67 by His leads to only a slight increase of the peroxidase activity in the Tyr67His/Met80Ala double mutant with respect to wild type, while the Tyr67His mutant behaves as wild type, as previously reported, suggests that the peroxidase activity of cytochrome c is linked to an overall conformational change of the heme pocket and not only to the disappearance of the Fe-Met80 bond. Therefore, in human cytochrome c there is an interplay between the two residues at positions 67 and 80 that affects the conformation of the distal side of the heme pocket, and thus the sixth coordination of the heme.
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.
Chiara
C
Ciaccio
Department of Chemistry "Ugo Schiff", University of Florence, Sesto Fiorentino, FI, Italy.
Lorenzo
L
Tognaccini
Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Rome, Italy.
Theo
T
Battista
Department of Sciences, University of Rome Tre, Rome, Italy.
Manuela
M
Cervelli
Department of Chemistry "Ugo Schiff", University of Florence, Sesto Fiorentino, FI, Italy.
Barry D
BD
Howes
Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Rome, Italy.
Roberto
R
Santucci
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.
Massimo
M
Coletta
Department of Sciences, University of Rome Tre, Rome, Italy.
Paolo
P
Mariottini
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.
Giulietta
G
Smulevich
Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Rome, Italy. Electronic address: fiorucci@uniroma2.it.
Laura
L
Fiorucci
D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006632:Histamine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D014443:Tyrosine; D006418:Heme; D006632:Histamine; D045304:Cytochromes c; D008715:Methionine; D000409:Alanine
Circular dichroism; Ligand binding kinetics; Peroxidase activity; Resonance Raman; Site directed mutagenesis