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Kinetics of Histidine Dissociation From the Heme Fe(III) in N-fragment (residues 1–56) of Cytochrome c

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Abstract

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 \rightleftharpoons 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.

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Abbreviations

cyt:

c

Cytochrome:

c

GuHCl:

Guanidine hydrochloride

HPLC:

High pressure liquid chromatography

SDS-PAGE:

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

TFA:

Trifluoroacetic acid

2-NBA:

2-nitrobenzaldehyde BG Bromocresol green

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Correspondence to Cristiano Viappiani.

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Abbruzzetti, S., Viappiani, C., Sinibaldi, F. et al. Kinetics of Histidine Dissociation From the Heme Fe(III) in N-fragment (residues 1–56) of Cytochrome c. Protein J 23, 519–527 (2004). https://doi.org/10.1007/s10930-004-7879-x

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  • DOI: https://doi.org/10.1007/s10930-004-7879-x

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