Direct electrochemical evidence for an equilibrium intermediate in the guanidine-induced unfolding of cytochrome c

Ferri, Tommaso; Poscia, Alessandro; Ascoli, Franca; Santucci, Roberto

doi:10.1016/s0167-4838(96)00122-7

Cited by 61 publications

(47 citation statements)

References 35 publications

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“…This suggests that the lack of the (57-104 residues) segment is a stabilizing factor for the N-fragment at acid pH as the remaining peptide shields better the heme from the solvent. These considerations are in very good agreement with the redox potentials (E 0 ' 250 mV (vs NHE)) for native cyt c, E 0 ' 10 mV for the GuHCl-unfolded cyt c, E 0 ' 0 mV for the N-fragment, at neutral pH and 25°C [16,37], that confirm the efficient shielding action of the 1-56 peptide in the N-fragment.…”

Section: Ferrous Formsupporting

confidence: 79%

A model for the misfolded bis-His intermediate of cytochrome c: the 1?56 N-fragment

Santoni

2004

Journal of Inorganic Biochemistry

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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.

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Section: Ferrous Formsupporting

confidence: 79%

A model for the misfolded bis-His intermediate of cytochrome c: the 1?56 N-fragment

Santoni

2004

Journal of Inorganic Biochemistry

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“…[26] The redox potential of À 0.425 V for B2[6cLS] compares well with the value for the bis-His ligated Cyt-c in urea at neutral pH values (À 0.4 V), [30] at which the heme pocket structure is assumed to be largely similar to that of B2[6cLS]. [21] The negative shift of the redox potential in B2[6cLS] compared to that in B1 is ascribed both to change in the ligation state and to the heme environment.…”

Section: Redox Potentialsmentioning

confidence: 55%

Redox and Conformational Equilibria and Dynamics of Cytochrome c at High Electric Fields

2003

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Cytochrome c (Cyt-c) adsorbed in the electrical double layer of the Ag electrode/electrolyte interface has been studied by stationary and time-resolved surface-enhanced resonance Raman spectroscopy to analyse the effect of strong electric fields on structure and reaction equilibria and dynamics of the protein. In the potential range between +0.1 and -0.55 V (versus saturated calomel electrode), the adsorbed Cyt-c forms a potential-dependent reversible equilibrium between the native state B1 and a conformational state B2. The redox potentials of the bis-histidine-coordinated six-coordinated low-spin and five-coordinated high-spin substates of B2 were determined to be -0.425 and -0.385 V, respectively, whereas the additional six-coordinated aquo-histidine-coordinated high-spin substate was found to be redox-inactive. The redox potential for the conformational state B1 was found to be the same as in solution in agreement with the structural identity of the adsorbed B1 and the native Cyt-c. For all three redox-active species, the formal heterogeneous electron transfer rate constants are small and of the same order of magnitude (3-13 s-1), which implies that the rate-limiting step is largely independent of the redox-site structure. These findings, as well as the slow and potential-dependent transitions between the various conformational (sub-)states, can be rationalized in terms of an electric field-induced increase of the activation energy for proton-transfer steps linked to protein structural reorganisation. Further increasing the electric field strength by shifting the electrode potential above +0.1 V leads to irreversible structural changes that are attributed to an unfolding of the polypeptide chain.

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“…For example, Myer 45 concluded that urea denaturation of horse heart cyt c is characterized by several optically monitored transitions implicating partially unfolded states. Especially clear evidence that an equilibrium intermediate accumulates prior to global unfolding of cyt c was presented by Ferri et al, 46 who found that protein species distinct from the native and the unfolded states, in terms of their redox properties, accumulate above 1 M GuHCl, within the region previously considered as the native baseline region. 44,47 Heme CD and absorbance measurements at 695 nm further confirmed the complex mechanism of cyt c equilibrium denaturation.…”

Section: Discussionmentioning

confidence: 94%

“…44,47 Heme CD and absorbance measurements at 695 nm further confirmed the complex mechanism of cyt c equilibrium denaturation. 46 Conventional and magnetic CD measurements in the Soret region were subsequently used to characterize reduced and oxidized forms of cyt c and provided additional support for the existence of the partially unfolded state below global unfolding region. 48 Another recent study made use of CD and heme absorbance to show non-coincidence of denaturation curves monitored at different wavelengths.…”

Section: Discussionmentioning

confidence: 99%

Structural Characterization of an Equilibrium Unfolding Intermediate in Cytochrome c

Latypov

Cheng

Roder

et al. 2006

Journal of Molecular Biology

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Although the denaturant-induced unfolding transition of cytochrome c was initially thought to be a cooperative process, recent spectroscopic studies have shown deviations from two-state behavior consistent with accumulation of an equilibrium intermediate. However, little is known about the structural and thermodynamic properties of this state, and whether it is stabilized by the presence of non-native heme ligands. We monitored the reversible denaturant-induced unfolding equilibrium of oxidized horse cytochrome c using various spectroscopic probes, including fluorescence, near and far-UV CD, heme absorbance bands in the Soret, visible and near-IR regions of the spectrum, as well as 2D NMR. Global fitting techniques were used for a quantitative interpretation of the results in terms of a three-state model, which enabled us to determine the intrinsic spectroscopic properties of the intermediate. A well-populated intermediate was observed in equilibrium experiments at pH 5 using either guanidine-HCl or urea as a denaturant, both for wild-type cytochrome c as well as an H33N mutant chosen to prevent formation of non-native Hisheme ligation. For a more detailed structural characterization of the intermediate, we used 2D 1 H-15 N correlation spectroscopy to follow the changes in peak intensity for individual backbone amide groups. The equilibrium state observed in our optical and NMR studies contains many native-like structural features, including a well-structured a-helical subdomain, a short Trp59-heme distance and solvent-shielded heme environment, but lacks the native Met80 sulfur-iron linkage and shows major perturbations in side-chain packing and other tertiary interactions. These structural properties are reminiscent of the A-state of cytochrome c, a compact denatured form found under acidic high-salt conditions, as well as a kinetic intermediate populated at a late stage of folding. The denaturant-induced intermediate also resembles alkaline forms of cytochrome c with altered heme ligation, suggesting that disruption of the native methionine ligand favors accumulation of structurally analogous states both in the presence and absence of non-native ligands.

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Direct electrochemical evidence for an equilibrium intermediate in the guanidine-induced unfolding of cytochrome c

Cited by 61 publications

References 35 publications

A model for the misfolded bis-His intermediate of cytochrome c: the 1?56 N-fragment

A model for the misfolded bis-His intermediate of cytochrome c: the 1?56 N-fragment

Redox and Conformational Equilibria and Dynamics of Cytochrome c at High Electric Fields

Structural Characterization of an Equilibrium Unfolding Intermediate in Cytochrome c

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