A model for the misfolded bis-His intermediate of cytochrome c: the 1-56 N-fragment.
We have characterized the ferric and ferrous forms of the heme-containing (1-56 residues) N-fragment of horse heart cytochrome c (cyt c) at different pH values and low ionic strength by UV-visible absorption and resonance Raman (RR) scattering. The results are compared with native cyt c in the same experimental conditions as this may provide a deeper insight into the cyt c unfolding-folding process. Folding of cyt c leads to a state having the heme iron coordinated to a histidine (His18) and a methionine (Met80) as axial ligands. At neutral pH the N-fragment (which lacks Met80) shows absorption and RR spectra that are consistent with the presence of a bis-His low spin heme, like several non-native forms of the parental protein. In particular, the optical spectra are identical to those of cyt c in the presence of a high concentration of denaturants; this renders the N-fragment a suitable model to study the heme pocket microenvironment of the misfolded (His-His) intermediate formed during folding of cyt c. Acid pH affects the ligation state in both cyt c and the N-fragment. Data obtained as a function of pH allow a correlation between the structural properties in the heme pocket of the N-fragment and those of non-native forms of cyt c. The results underline that the (57-104 residues) segment under native-like conditions imparts structural stability to the protein by impeding solvent access into the heme pocket.
Santoni
Elisa
E
Dipartimento di Chimica, Università di Firenze, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Florence, Italy.
Scatragli
Silvia
S
Sinibaldi
Federica
F
Fiorucci
Laura
L
Santucci
Roberto
R
Smulevich
Giulietta
G
D000818:Animals; D045304:Cytochromes c / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006736:Horses; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009206:Myocardium / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D010455:Peptides; D006418:Heme; D006639:Histidine; D045304:Cytochromes c