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

Santoni, Elisa

doi:10.1016/s0162-0134(04)00062-5

Cited by 12 publications

(37 citation statements)

References 24 publications

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“…Recently, analysis of the low frequency region (390–420 cm −1 ) of the RR spectrum of a noncovalent complex reconstituted upon mixing two noncontiguous fragments of cyt c proved to be decisive in distinguishing between a Met, Lys, or His ligand bound to the heme iron (Caroppi et al 2004). In the present case, the changes in intensity and frequency of the δ (C β C a S) band at 396 cm −1 and the δ (C β C a C b ) band at 413 cm −1 upon addition of oleic acid to cyt c are not consistent with the changes observed when cyt c adopts bis‐His coordination (Santoni et al 2004), where a significant intensity increase of a δ (C β C a C b ) band at 418 cm −1 is expected. On the contrary, they are similar to those observed following the alkaline transition of cyt c (Dopner et al 1999), characterized by two conformers, in which Lys73 or Lys79 replaces the axial Met ligand bound to the heme iron at neutral pH.…”

Section: Resultscontrasting

confidence: 79%

ATP specifically drives refolding of non‐native conformations of cytochrome c

Sinibaldi

2005

Protein Science

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An increasing body of evidence ascribes to misfolded forms of cytochrome c (cyt c) a role in pathophysiological events such as apoptosis and disease. Here, we examine the conformational changes induced by lipid binding to horse heart cyt c at pH 7 and study the ability of ATP (and other nucleotides) to refold several forms of unfolded cyt c such as oleic acid-bound cyt c, nicked cyt c, and acid denatured cyt c. The CD and fluorescence spectra demonstrate that cyt c unfolded by oleic acid has an intact secondary structure, and a disrupted tertiary structure and heme environment. Furthermore, evidence from the Soret CD, electronic absorption, and resonance Raman spectra indicates the presence of an equilibrium of at least two low-spin species having distinct heme-iron(III) coordination. As a whole, the data indicate that binding of cyt c to oleic acid leads to a partially unfolded conformation of the protein, resembling that typical of the molten globule state. Interestingly, the native conformation is almost fully recovered in the presence of ATP or dATP, while other nucleotides, such as GTP, are ineffective. Molecular modeling of ATP binding to cyt c and mutagenesis experiments show the interactions of phosphate groups with Lys88 and Arg91, with adenosine ring interaction with Glu62 explaining the unfavorable binding of GTP. The finding that ATP and dATP are unique among the nucleotides in being able to turn non-native states of cyt c back to native conformation is discussed in the light of cyt c involvement in cell apoptosis.

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Section: Resultscontrasting

confidence: 79%

ATP specifically drives refolding of non‐native conformations of cytochrome c

Sinibaldi

2005

Protein Science

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“…Consequently, many changes are observed in the electronic absorption and RR spectra. The common spectroscopic markers of all misligated 6cLS forms are (a) a blueshifted Soret band, (b) loss of the CT band at 695 nm in the visible region of the electronic absorption spectrum; (c) the upshifting of the core size marker bands with respect to those of the WT; (d) a rather simple low‐wavenumber RR spectrum because the out‐of‐plane modes become weak or disappear . The most dramatic changes are observed in the fingerprint region, where specific iron‐ligand stretching vibrations corresponding to His18–Fe–His and His18–Fe–Lys misligation as well as a specific band pattern for His18–Fe–OH − and His18–Fe–H 2 O conformations are observed (see below).…”

Section: Resultsmentioning

confidence: 99%

“…The most dramatic changes are observed in the fingerprint region, where specific iron‐ligand stretching vibrations corresponding to His18–Fe–His and His18–Fe–Lys misligation as well as a specific band pattern for His18–Fe–OH − and His18–Fe–H 2 O conformations are observed (see below). Therefore, this spectral region will be described in detail for each model compound, because it provides a useful marker for the identification of the non‐native conformations …”

Section: Resultsmentioning

confidence: 99%

“…The overall spectral changes in the fingerprint region (Figure , right) that follow the formation of the 6cLS His18–Fe–His/Im species reported for (a) the Cyt c imidazole model compound, (b) the His18–Fe–His (bis‐His) coordinated N‐fragment of Cyt c, and (c) the imidazole and imidazolate complexes of microperoxidase are a blueshift of the ν 8 band to 344 cm −1 and an increase in the intensity of the δ(C β C a C b ) band at 418 cm −1 . Moreover, a new band appears at 400 cm −1 , which is assigned to the ν asym (Fe‐Im 2 ) mode.…”

Section: Resultsmentioning

confidence: 99%

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Probing the non‐native states of Cytochrome c with resonance Raman spectroscopy: A tool for investigating the structure–function relationship

Milazzo

Tognaccini

Howes

et al. 2018

J Raman Spectroscopy

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Cytochrome c (Cyt c) is a single polypeptide chain hemoprotein located in the mitochondrial intermembrane space. Under physiological conditions, His18 and Met80 are the fifth and sixth axial ligands, respectively, of the heme iron. Cyt c plays important roles in the cell because it acts as an electron carrier in the respiratory chain and as a reactive oxygen species scavenger. Moreover, direct interaction with cardiolipin, a phospholipid of the mitochondrial membrane, induces acquisition of peroxidase activity and subsequent release of Cyt c into the cytosol, where it acts as an apoptosis initiator. Cyt c has been extensively studied as a model protein to understand the general principles of protein folding, and it has been reported that the Met80 sixth ligand can be replaced by other internal or exogenous ligands depending on the pH or the presence of denaturing agents. The combination of ultraviolet–visible absorption and resonance Raman (RR) spectroscopy is a precious tool to identify the sixth heme iron ligand in the misligated forms. In particular, specific RR markers have been identified for each non‐native ligand. In this review, we illustrate the spectral variations and the corresponding RR marker bands observed for the different non‐native species. Furthermore, on the basis of these findings, we discuss the results obtained on the interaction of Cyt c with cardiolipin and the effect of mutating key residues in the Cyt c heme cavity, which has enhanced insight into protein unfolding and apoptosis.

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“…In an attempt to possibly determine the amino acid side-chain residues of fragment 1-44 that act as ligands for the heme iron, resonance Raman (RR) measurements on the heme-fragment complex were carried out using an excitation wavelength in the Soret band at 413.1 nm (47). The high (ν 4 , ν 3 , ν 2 , ν 10 at 1373, 1504, 1576, and 1639 cm -1 , respectively) and low (ν 9 and ν 8 at 268 and 346 cm -1 ) frequency region spectra of the complex showed frequencies typical of an Fe(III) in a hexacoordinated low spin state (not shown) (see ref 25).…”

Section: Resultsmentioning

confidence: 99%

Heme Binding by the N-Terminal Fragment 1−44 of Human Growth Hormone

2005

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Fragment 1-44 of human growth hormone (hGH), prepared in vitro by limited proteolysis of the hormone with pepsin at low pH, encompasses in full the N-terminal helix of this four-helix bundle protein [Spolaore, B., Polverino de Laureto, P., Zambonin, M., and Fontana, A. (2004) Biochemistry 40, 9460-9468]. Here, we report the new and interesting observation that fragment 1-44 can bind heme. The binding property is specific for the N-terminal helix of hGH, since heme binding does not occur with fragment 45-191 or the entire protein. The spectral characteristics of Fe-protoporphyrin IX are those of a low-spin, hexacoordinated iron ligated by two imidazole rings of His residues or His and Met residues. Far-UV circular dichroism (CD) measurements revealed that fragment 1-44 acquires a helical secondary structure upon heme binding. Heme appears to be bound to the fragment in a stereospecific way, since an induced dichroic signal is observed in the Soret region of the CD spectrum. The heme-fragment complex occurs in a 1:1 molar ratio, as determined by spectrophotometric titration, as well as by electrospray-ionization mass spectrometric analysis of the complex. The fragment alone is much more susceptible to tryptic digestion than the heme complex, implying a more folded and rigid structure of this last species. It is proposed that the molecular features of fragment 1-44 determining its heme-binding property reside in the amphipathic character of the helix adopted by the fragment, as well as in the presence in its polypeptide chain of His18, His21, and Met14. These residues can act as specific ligands for the heme-iron, as observed with cytochromes.

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

Cited by 12 publications

References 24 publications

ATP specifically drives refolding of non‐native conformations of cytochrome c

ATP specifically drives refolding of non‐native conformations of cytochrome c

Probing the non‐native states of Cytochrome c with resonance Raman spectroscopy: A tool for investigating the structure–function relationship

Heme Binding by the N-Terminal Fragment 1−44 of Human Growth Hormone

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