Participation of the protein ligands in the folding of cytochrome c

Methylophilus methylotrophus cytochrome c" is an unusual monohaem protein (1 5 kDa) undergoing a redox-linked spin-state transition [Santos, H. & Turner, D. L. (1988) Biochim. Biophys. Actu 954,277 -2861. The midpoint redox potential of cytochrome c" was measured over the pH range 4-10. The pH dependence of the midpoint redox potential was interpreted in terms of a model that considers the redox-state dependence of the ionization of two distinct and non-interacting protonated groups in the protein. This analysis lcd to the following pK, values within the pH range studied: p e l = 6.4, p K l = 5.4 and p e 2 = 8.1. Proton-NMR spectroscopy was used to assist the characterization of the two ionizing groups responsible for the observed redox-Bohr effect : the group ionizing with a lower pPa was assigned to a haem propionic acid substituent and the other to the axial histidine ligand which becomes detached upon reduction, which has a pK", too low to be measured. It is shown that M . methylotrophus cytochrome cfr is able to couple electron and proton transfer in the physiological pH range through a mechanism involving reversible change in the haemiron coordination. Possible implications for the physiological role of the protein are discussed.Cytochrome c" is an unusual soluble monohaem cytochrome (15 kDa) present in the obligate methylotroph Methylophilus methylotrophus. The sequence of 44 residues in the N-terminus of the protein is known, but no significant degree of similarity has been found with the sequences of other proteins [l]. NMR and ultraviolet/visible spectroscopies have shown that the haem undergoes a redox-linked spin-state transition, going from a low-spin state in the oxidized form to a high-spin state in the reduced form. The axial ligands were found to be two histidine residues in the oxidized form by magnetic CD [2] and a single histidine in the reduced form by NMR [l]. Furthermore, magnetic CD and EPR provided strong evidence for a near-perpendicular orientation of the two axial histidine residues in the oxidized form [2].Ferricytochrome c" remains as a unique example of a haem-c protein with bis-histidine coordination, displaying spectroscopic features which are similar to those found in model compounds where the axial ligand planes are forced into a perpendicular orientation by steric constraints [ 3 , 41.Cytochrome c" binds CO in the reduced form, and both oxidized and reduced forms bind cyanide but only at very high pH (> 10) [l]. At pH > 30, the protein undergoes a major conformational change and becomes low spin in both oxidation states [l J, but this non-physiological alkaline transition will not be dealt with in the present article. Earlier NMR studies of cytochrome c" have shown that some of the hyperfine-shifted resonances present a strong pH dependence in the pH region 4-9 [l], some of the haem methyl resonances in the oxidized form experiencing a shift as large as 3 ppm. This pH dependence is unusually large for a low-spin cytochrome and led us to expect a pH dependence of the midpoint ...

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“…pK, < < 2.5 agrees well with the pK, < 3 reported for histidines when coordinated to iron [21]. As the inset in Fig.…”

Section: Discussionsupporting

confidence: 88%

Involvement of a labile axial histidine in coupling electron and proton transfer in Methylophilus methylotrophus cytochrome c″

Costa

Santos

Turner

et al. 1992

European Journal of Biochemistry

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“…21,58 Studies of oxidized cyt c from several species have shown that the native methionine sulfur-iron ligand is replaced by a His, Lys or N-terminal NH 2 (if unprotected) under denaturing conditions near neutral pH or above. 30,35,38 The presence of a non-native heme ligand can result in the trapping of transient folding intermediates if folding experiments are conducted near neutral pH.…”

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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“…The pH was adjusted to 7 by addition of ammonia. UV-Vis measurements ( Soret ϭ 1.06 ϫ 10 5 M Ϫ1 cm Ϫ1 [63]) indicate the loss of roughly 30% protein during the procedure, yielding a final stock solution of about 35 M. Native cyt c samples were prepared in 5 mM ammonium acetate at pH 7 and acid-denatured protein was generated by addition of formic acid to pH 2 (both without heating). Protein digestion was performed by trypsin spin columns (Sigma).…”

Section: Methodsmentioning

confidence: 99%

“…The UV-Vis absorption spectrum reports on the heme environment [11,63]. Coordination of the heme iron with its native His18 and Met80 ligands leads to a Soret absorption maximum at 409 nm.…”

Section: Optical Spectroscopymentioning

confidence: 99%

Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

Liu

Konermann

2009

J. Am. Soc. Mass Spectrom.

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This work uses electrospray ionization mass spectrometry (ESI-MS) in conjunction with hydrogen/deuterium exchange (HDX) and optical spectroscopy for characterizing the solutionphase properties of cytochrome c (cyt c) after heat exposure. Previous work demonstrated that heating results in irreversible denaturation for a subpopulation of proteins in the sample. However, that study did not investigate the physical reasons underlying this interesting effect. Here we report that the formation of oxidative modifications at elevated temperature plays a key role for the observed behavior. Tryptic digestion followed by tandem mass spectrometry is used to identify individual oxidation sites. Trp59 and Met80 are among the modified amino acids. In native cyt c both of these residues are buried deep within the protein structure, such that covalent modifications would be expected to be particularly disruptive. ESI-MS analysis after heat exposure results in a bimodal charge-state distribution. Oxidized protein appears predominantly in charge states around 11ϩ, whereas a considerably lower degree of oxidation is observed for the 7ϩ and 8ϩ peaks. This finding confirms that different oxidation levels are associated with different solution-phase conformations. HDX measurements for different charge states are complicated by peak distortions arising from oxygen adduction. Nonetheless, comparison with simulated peak shapes clearly shows that the HDX properties are different for high-and low-charge states, confirming that interconversion between unfolded and folded conformers is blocked in solution. In addition to oxidation, partial aggregation upon heat exposure likely contributes to the formation of irreversibly denatured protein. A number of well-established tools are available for experiments of this kind, including X-ray crystallography, nuclear magnetic resonance, calorimetry, and various spectroscopic techniques. Electrospray ionization mass spectrometry (ESI-MS) has become another widely used approach for exploring the properties of proteins in solution, providing information complementary to that obtained by traditional methods [1]. The ESI process generates intact gas-phase ions from proteins in solution. In the commonly used positive-ion mode these [M ϩ nH] nϩ species are multiply charged because of proton attachment.ESI of natively folded proteins results in protonation states n similar to those predicted for water droplets of the same size that are charged to the Rayleigh limit. This finding supports the idea that ionization follows the charged-residue model [2-6], although alternative scenarios have been proposed as well [7][8][9]. Much higher protonation states and wider charge-state distributions are generally seen for proteins that are unfolded in solution. Based on this empirical relationship, ESI-MS is now routinely being used for monitoring structural transitions in response to changes in pH, temperature, organic co-solvents, or covalent modifications [1, 10 -16]. The physical basis for the striking dependence of...

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Participation of the protein ligands in the folding of cytochrome c

Cited by 191 publications

References 19 publications

Involvement of a labile axial histidine in coupling electron and proton transfer in Methylophilus methylotrophus cytochrome c″

Involvement of a labile axial histidine in coupling electron and proton transfer in Methylophilus methylotrophus cytochrome c″

Structural Characterization of an Equilibrium Unfolding Intermediate in Cytochrome c

Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

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