Kinetics of histidine dissociation from the heme Fe(III) in N-fragment (residues 1-56) of cytochrome c.
We have here investigated the dissociation kinetics of the His side chains axially ligated to the heme-iron in the ferric (1-56 residues) N-fragment of horse cyt c. The ligand deligation induced by acidic pH-jump occurs as a biexponential process with different pre-exponential factors, consistent with a structural heterogeneity in solution and the presence of two differently coordinated species. In analogy with GuHCl-denatured cyt c, our data indicate the presence in solution of two ferric forms of the N-fragment characterized by bis-His coordination, as summarized in the following scheme: His18-Fe(III)-His26 <==> His18-Fe(III)-His33. We have found that the pre-exponential factors depend on the extent of the pH-jump. This may be correlated with the different pKa values shown by His26 and His33; due to steric factors, His26 binds to the heme-Fe(III) less strongly than His33, as recently shown by studies on denatured cyt c. Interestingly, the two lifetimes are affected by temperature but not by the extent of the pH-jump. The lower pKa for the deligation reaction required the use of an improved laser pH-jump setup, capable of inducing changes in H+ concentration as large as 1 mM after the end of the laser pulse. For the ferric N-fragment, close activation entropy values have been determined for the two histidines coordinated to the iron; this result significantly differs from that for GuHCl-denatured cyt c, where largely different values of activation entropy were calculated. This underlines the role played by the missing segment (residues 57-104) peptide chain in discriminating deligation of the "nonnative" His from the sixth coordination position of the metal.
Abbruzzetti
Stefania
S
Dipartimento di Fisica, Università degli Studi di Parma, Parco Area delle Scienze 7/A 43100 Parma, Italy.
Viappiani
Cristiano
C
Sinibaldi
Federica
F
Santucci
Roberto
R
D000818:Animals; D045304:Cytochromes c / Q000737:chemistry*; D004789:Enzyme Activation; D006639:Histidine / Q000737:chemistry*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007834:Lasers; D008024:Ligands; D010449:Peptide Mapping; D010782:Photolysis; D013053:Spectrophotometry
D008024:Ligands; D006639:Histidine; D045304:Cytochromes c