Stability of the Heme Fe−N-Terminal Amino Group Coordination Bond in Denatured Cytochrome c

Abstract

In the denatured states of Hydrogenobacter thermophilus cytochrome c(552) (HT) and Pseudomonas aeruginosa cytochrome c(551) (PA), and their mutants, the N-terminal amino group of the polypeptide chain is coordinated to heme Fe in place of the axial Met, the His-N(term) form being formed. The coordination of the N-terminal amino group to heme Fe leads to loop formation by the N-terminal stretch preceding the first Cys residue bound to the heme, and the N-terminal stretches of HT and PA are different from each other in terms of both the sequence and the number of constituent amino acid residues. The His-N(term) form was shown to be rather stable, and hence it can influence the stability of the denatured state. We have investigated the heme Fe coordination structures and stabilities of the His-N(term) forms emerging upon guanidine hydrochloric acid-induced unfolding of the oxidized forms of the proteins. The Fe-N(term) coordination bond in the His-N(term) form with a 9-residue N-terminal stretch of HT proteins was found to be tilted to some extent away from the heme normal, as reflected by the great heme methyl proton shift spread. On the other hand, the small heme methyl proton shift spread of the His-N(term) form with an 11-residue stretch of PA proteins indicated that its Fe-N(term) bond is nearly parallel with the heme normal. The stability of the His-N(term) form was found to be affected by the structural properties of the N-terminal stretch, such as its length and the N-terminal residue. With a given N-terminal residue, the stability of the His-N(term) form is higher for a 9-residue N-terminal stretch than an 11-residue one. In addition, with a given length of the N-terminal stretch, the His-N(term) form with an N-terminal Glu is stabilized by a few kJ mol(-1) relative to that with an N-terminal Asn. These results provide a novel insight into the stabilizing interactions in the denatured cyts c that will facilitate elucidation of the folding/unfolding mechanisms of the proteins.

Control of the Stability of Hydrogenobacter Thermophilus Cytochrome c552 through Alteration of the Basicity of the N-Terminal Amino Group of the Polypeptide Chain

“…We next compared the heme electronic structures of the oxidized forms of the His−N term forms of the mutant proteins with that of the wild-type HT through analysis of their 1 H NMR spectra (Figure ). As has been demonstrated previously, the heme methyl proton signals of the His−N term form of the protein, observed in 15−40 ppm, were slightly affected by the replacement of the N-terminal amino acid residue . In addition, the signals emerging in the shift range characteristic of the HS ferriheme species, , that is, downfield of 50 ppm, were observed for the all denatured proteins at p 2 H 7.0, and their intensities varied among the proteins (see Supporting Information).…”

“…In the 1 H NMR spectra of the oxidized cyts c , the resolved heme methyl and Fe-bound Met proton signals, which are highly sensitive to the heme active site structure, − were essentially unaltered by the amino acid replacements (see Supporting Information). These results indicated that, as in the cases of the mutant proteins previously studied, the heme active site structure is not affected by the replacement of the N-terminal amino acid residue …”

“…Absorption spectra of 5 μM proteins were recorded on a Beckman DU 640 spectrophotometer. Cleavage of the Fe−N term bond of the His−N term form with decreasing pH was monitored at 25 °C as the pH dependence of the 399.5 nm absorbance ( A 399.5 ) (see Supporting Information), and the results were fitted to the following equation to yield the p K 3 value, A 399.5 = false{ A 399.5 , LS + A 399.5 , HS false[ 10 n false( normalp K 3 − pH false) false] false} / false[ 1 + 10 n false( normalp K 3 − pH false) false] where A 399.5,LS and A 399.5,HS are the 399.5 nm absorbance of the LS His−N term form and the HS ferriheme species resulting from the loss of the Fe−N term bond, respectively, and n is the number of protons involved in the process. But since the K 2 value of the proteins considered in the study are relatively small (see below), deprotonated high spin form emerges almost throughout the course of the pH titration for determining the p K 3 values of proteins.…”

“…It is now widely accepted that the denatured states of proteins can contain somewhat ordered structures known as residual structures. − Such residual structures can significantly influence the energy of a denatured state . We have shown that a residual structure is formed upon guanidine hydrochloric acid (GdnHCl)-induced unfolding of the oxidized form of Hydrogenobacter thermophilus cytochrome c 552 (HT) . HT is a class I cytochrome c (cyt c ) that contains a single heme covalently bound to a polypeptide chain composed of 80 amino acid residues. , In HT, His and Met residues are coordinated to the heme Fe as axial ligands in the native state. , However, upon unfolding of the protein, the N-terminal amino group of the polypeptide chain acts as an axial ligand for the heme Fe in place of the native axial Met, the His−N term form being formed. , Since the formation of the His−N term form has been shown to significantly influence the energy of the denatured state, detailed knowledge of the properties of the His−N term form is useful for manipulation of the Δ G ND value through the energy of the denatured state.…”

“…The bond between heme Fe and the N-terminal amino group (Fe−N term bond) is broken as the N-terminal amino group is protonated under low pH conditions. Since cleavage of the Fe−N term bond accompanies a change in the spin state of the heme Fe, that is, the ferric low spin (LS) His−N term form is converted to a high spin (HS) ferriheme species, ,− this process can be readily and sensitively detected through observation of the Soret absorption change, and hence equilibrium constant K 3 in Scheme is yielded by fitting of the data to the Henderson−Hasselbalch equation. ,, This equilibrium can be represented by a two-step process, that is, deprotonation of the N-terminal amino group and formation of the Fe−N term bond, which can be assessed on the basis of equilibrium constants K 1 and K 2 , respectively. Since K 1 can be estimated through the study of an appropriate model peptide, the K 2 value, which reflects the thermodynamic stability of the His−N term form, can be obtained as K 2 = K 3 / K 1 , if K 2 ≫ 1, or on numerical fitting, if otherwise (see Materials and Methods).…”