The Conformational Flexibility of Oxidized Cytochrome c Studied through Its Interaction with NH3 and at High Temperatures

Banci, Lucia; Bertini, Ivano; Spyroulias, Georgios A.; Turano, Paola

doi:10.1002/(sici)1099-0682(199805)1998:5<583::aid-ejic583>3.0.co;2-y

Cited by 39 publications

(48 citation statements)

References 82 publications

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“…When the native Met80 heme ligand is exchanged for another proteindonated (e.g., histidine or lysine) [15,21] or exogenous ligand [32,33,34,35,36], the chemical shifts of the heme methyl protons change dramatically. For example, the alkaline form of h-cyt c, in which the Met80 ligand is replaced by deprotonated Lys side chains, is readily identified by NMR spectroscopy (Fig.…”

Section: Detection Of Non-native Forms Of H-cyt C In Guhclmentioning

confidence: 99%

“…Conformers of h-cyt c in which one or more Lys bind heme in place of Met also have been identified at neutral pH in the presence of denaturants in this work and in previous studies [15], and also as intermediates on the folding pathway [51]. Furthermore, Lys binding to the h-cyt c heme has been detected in the presence of nitrogenous bases such as imidazole [53], in the presence of acetonitrile [41], and at elevated temperatures (55°C for h-cyt c; 30°C below denaturation temperature) [36]. It seems likely that these states have some relevance for cyt c function, given that they are populated under a variety of conditions.…”

Section: Comparison Of Urea and Guhcl Denatured Proteinsmentioning

confidence: 99%

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Denaturant dependence of equilibrium unfolding intermediates and denatured state structure of horse ferricytochrome c

Russell

Bren

2002

J Biol Inorg Chem

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Nuclear magnetic resonance spectroscopy is employed to characterize unfolding intermediates and the denatured state of horse ferricytochrome c in guanidine hydrochloride. Unfolded and partially unfolded species with non-native heme ligation are detected by analysis of hyperfine-shifted (1)H resonances. Two equilibrium unfolding intermediates with His-Lys heme axial ligation are detected, as are two unfolded species with bis-His heme ligation. These results are contrasted with previous results on horse ferricytochrome c denaturation by urea, for which only one unfolding intermediate and one unfolded species were detected by NMR spectroscopy. Urea and guanidine hydrochloride are often used interchangeably in protein denaturation studies, but these results and those of others indicate that unfolded and intermediate states in these two denaturants may have substantially different properties. Implications of these results for folding studies and the biological function of mitochondrial cytochromes c are discussed.

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Section: Detection Of Non-native Forms Of H-cyt C In Guhclmentioning

confidence: 99%

Section: Comparison Of Urea and Guhcl Denatured Proteinsmentioning

confidence: 99%

Denaturant dependence of equilibrium unfolding intermediates and denatured state structure of horse ferricytochrome c

Russell

Bren

2002

J Biol Inorg Chem

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“…Structural information has been acquired only very recently for the alkaline conformer [18], demonstrating the importance of the topological connections between the aforementioned additional short helices and surrounding residues (especially residues on the loop bearing Met80) to maintain the native structure. Release of the native axial ligand and substitution with a lysine also occur at high temperature (the pK a of the alkaline transition being strongly temperature-dependent) [19,20], events that also have been proposed to have a role in denaturant-induced cytochrome c unfolding [21,22]. Stopped flow H/D exchange data indicate that the events giving rise to the alkaline transition are controlled by the same structural unfolding reactions that account for the first steps of the unfolding pathway of cytochrome c [23], providing a kinetic picture where both the alkaline transition and the unfolding process can be described in terms of folding units of different stability.…”

Section: Introductionmentioning

confidence: 99%

The stability of the cytochrome c scaffold as revealed by NMR spectroscopy

Berners‐Price¹,

Bertini²,

Gray³

et al. 2004

Journal of Inorganic Biochemistry

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“…The detachment of the Met ligand from iron coordination is the first step of cytochrome c unfolding observed on increasing pH and/or temperature (Elöve et al 1994;Banci et al 1998). The pH-induced protein conformational transitions and changes in the ligation state of the heme iron in cytochrome c 2 from Rps.…”

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confidence: 97%

Cleavage of the iron-methionine bond in c-type cytochromes: Crystal structure of oxidized and reduced cytochrome c2 from Rhodopseudomonas palustris and its ammonia complex

et al. 2002

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The three-dimensional structures of the native cytochrome c 2 from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidized state at 1.70 Å and in the reduced state at 1.95 Å resolution. These are the first high-resolution crystal structures in both oxidation states of a cytochrome c 2 with relatively high redox potential (+350 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The unusual six-residue insertion Gly82-Ala87, which precedes the heme binding Met93, forms an isolated 3 10 -helix secondary structural element not previously observed in other c-type cytochromes. Furthermore, this cytochrome shows an external methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c 2 with other c-type cytochromes has revealed that the presence of such a residue, with torsion angles and of approximately −140 and −130°, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 Å resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X-ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localized change in backbone conformation, involving mainly the residues Lys92, Met93, and Thr94. Previous studies under typical denaturing conditions, including high-pH values and the presence of exogenous ligands, have shown that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c-type cytochromes. The ammonia adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H-bond between the Met93 and Tyr66 residues that stabilizes the native form, and the presence in this bacterial cytochrome c 2 of the rare six-residue insertion in the helix 3 10 conformation that increases Met loop flexibility.

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The Conformational Flexibility of Oxidized Cytochrome c Studied through Its Interaction with NH3 and at High Temperatures

Cited by 39 publications

References 82 publications

Denaturant dependence of equilibrium unfolding intermediates and denatured state structure of horse ferricytochrome c

Denaturant dependence of equilibrium unfolding intermediates and denatured state structure of horse ferricytochrome c

The stability of the cytochrome c scaffold as revealed by NMR spectroscopy

Cleavage of the iron-methionine bond in c-type cytochromes: Crystal structure of oxidized and reduced cytochrome c2 from Rhodopseudomonas palustris and its ammonia complex

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