Role of heme axial ligands in the conformational stability of the native and molten globule states of horse cytochrome c.


Abstract

One unique aspect of cytochrome c folding concerns the involvement of the covalently attached heme group and its axial ligands. To elucidate the role of the ligands in stabilizing the native and molten globule states, we studied the conformational and thermodynamic features of the iron-free derivative of horse cyctochrome c (porphyrin-cytochrome c). At neutral pH, far-UV circular dichroism suggested that porphyrin-cytochrome c has native-like alpha-helices, whereas near-UV CD suggested that the side-chains are flexible. Its stability against heat or denaturants was much less than that of the intact protein, and similar to that of the acidic molten globule state of the holoprotein. These results indicate that, at neutral pH, the ligation of His18 of the iron is important for the maintenance of the native structure whereas the Met80 ligation is not essential, and that porphyrin-cytochrome c assumes a molten globule-like state. Porphyrin-cytochrome c was largely unfolded at pH 2.0 in the absence of salt, but assumed another molten globule-like structure in the presence of anions. The salt-induced stabilization of the molten globule-like state was the same as that of apocytochrome c, requiring a much higher salt concentration than holocytochrome c. These results indicate that, at acidic pH, the His18 ligation is important, although not essential, for stabilizing the molten globule state. Taken together, both specific (i.e. the His18 axial ligand, as observed at acidic pH) and nonspecific interactions (the hydrophobic effects of the heme, as observed at neutral pH) contribute to stabilizing the molten globule state. Study holds ProTherm entries: 8897, 8898, 8899, 8900, 8901, 8902, 8903, 8904, 8905, 8906, 8907, 8908, 8909 Extra Details: cytochrome c; heme; molten globule; porphyrin-cytochrome c;,protein folding

Submission Details

ID: j3eeqCQ43

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:37 p.m.

Version: 1

Publication Details
Hamada D;Kuroda Y;Kataoka M;Aimoto S;Yoshimura T;Goto Y,J. Mol. Biol. (1996) Role of heme axial ligands in the conformational stability of the native and molten globule states of horse cytochrome c. PMID:8609608
Additional Information

Study Summary

Number of data points 29
Proteins Cytochrome c ; Cytochrome c
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: Cm buffers:glycine-HCl: 5-10 mM, pH:2.0, ionic:NaClO4: 200 mM ; Experimental Assay: m buffers:glycine-HCl: 5-10 mM, pH:2.0, ionic:NaClO4: 200 mM ; Experimental Assay: dG_H2O buffers:glycine-HCl: 5-10 mM, pH:2.0, ionic:NaClO4: 200 mM ; Experimental Assay: Cm ionic:NaCl: , buffers:Sodium phosphate: 5-10 mM, pH:7.0 ; Experimental Assay: m ionic:NaCl: , buffers:Sodium phosphate: 5-10 mM, pH:7.0 ; Experimental Assay: dG_H2O ionic:NaCl: , buffers:Sodium phosphate: 5-10 mM, pH:7.0 ; Experimental Assay: Cm buffers:Sodium phosphate: 5-10 mM, ionic:NaCl: , pH:7.0 ; Experimental Assay: m buffers:Sodium phosphate: 5-10 mM, ionic:NaCl: , pH:7.0 ; Experimental Assay: dG_H2O buffers:Sodium phosphate: 5-10 mM, ionic:NaCl: , pH:7.0 ; Experimental Assay: dG pH:3.1, prot_conc:0.1-1 mg/ml, buffers:Sodium acetate: 5-10 mM ; Experimental Assay: dG prot_conc:0.1-1 mg/ml, buffers:Sodium acetate: 5-10 mM, pH:3.4 ; Experimental Assay: dG pH:3.9, prot_conc:0.1-1 mg/ml, buffers:Sodium acetate: 5-10 mM ; Experimental Assay: dCp ; Experimental Assay: dHvH ; Experimental Assay: dG prot_conc:0.1-1 mg/ml, pH:4.8, buffers:Sodium acetate: 5-10 mM ; Experimental Assay: dG pH:6.0, prot_conc:0.1-1 mg/ml, buffers:Sodium phosphate: 5-10 mM ; Experimental Assay: dG buffers:Sodium phosphate: 5-10 mM, prot_conc:25 mM, pH:7.0
Libraries Mutations for sequence GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFTYTDANKNKGITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1LC2 2003-06-03 Solution Structure Of Reduced Horse Heart Cytochrome c in 30% Acetonitrile Solution, NMR 30 Structures
1LC1 2003-06-03 Solution Structure Of Reduced Horse Heart Cytochrome c in 30% Acetonitrile Solution, NMR Minimized Average Structure
2N3B 2015-10-28 Structure of oxidized horse heart cytochrome c encapsulated in reverse micelles
1FI7 2000-08-23 Solution structure of the imidazole complex of cytochrome C
5ZKV 2019-05-22 Solution structure of molten globule state of L94G mutant of horse cytochrome-c
1GIW 1998-12-09 SOLUTION STRUCTURE OF REDUCED HORSE HEART CYTOCHROME C, NMR, MINIMIZED AVERAGE STRUCTURE
1I5T 2001-03-21 SOLUTION STRUCTURE OF CYANOFERRICYTOCHROME C
1FI9 2000-08-23 SOLUTION STRUCTURE OF THE IMIDAZOLE COMPLEX OF CYTOCHROME C
2FRC 1997-07-29 CYTOCHROME C (REDUCED) FROM EQUUS CABALLUS, NMR, MINIMIZED AVERAGE STRUCTURE
1AKK 1997-09-17 SOLUTION STRUCTURE OF OXIDIZED HORSE HEART CYTOCHROME C, NMR, MINIMIZED AVERAGE STRUCTURE
2GIW 1998-12-09 SOLUTION STRUCTURE OF REDUCED HORSE HEART CYTOCHROME C, NMR, 40 STRUCTURES
1OCD 1997-06-16 CYTOCHROME C (OXIDIZED) FROM EQUUS CABALLUS, NMR, MINIMIZED AVERAGE STRUCTURE
1M60 2002-08-07 Solution Structure of Zinc-substituted cytochrome c
5C0Z 2016-09-21 1.12 The structure of oxidized rat cytochrome c at 1.13 angstroms resolution
5DF5 2016-09-14 1.3 The structure of oxidized rat cytochrome c (T28E) at 1.30 angstroms resolution.
5C9M 2016-09-21 1.36 The structure of oxidized rat cytochrome c (T28A) at 1.362 angstroms resolution.
2B4Z 2005-10-11 1.5 Crystal structure of cytochrome C from bovine heart at 1.5 A resolution.
6FF5 2018-03-21 1.74 X-ray structure of bovine heart cytochrome c at high ionic strength
3O1Y 2012-01-25 1.75 Electron transfer complexes: Experimental mapping of the redox-dependent cytochrome c electrostatic surface
3WUI 2014-07-16 1.8 Dimeric horse cytochrome c formed by refolding from molten globule state
3WC8 2013-12-11 1.8 Dimeric horse cytochrome c obtained by refolding with desalting method
1WEJ 1998-12-09 1.8 IGG1 FAB FRAGMENT (OF E8 ANTIBODY) COMPLEXED WITH HORSE CYTOCHROME C AT 1.8 A RESOLUTION
1HRC 1994-11-01 1.9 HIGH-RESOLUTION THREE-DIMENSIONAL STRUCTURE OF HORSE HEART CYTOCHROME C
3O20 2012-01-25 1.9 Electron transfer complexes:experimental mapping of the Redox-dependent Cytochrome C electrostatic surface
2YBB 2011-10-19 19.0 Fitted model for bovine mitochondrial supercomplex I1III2IV1 by single particle cryo-EM (EMD-1876)
5IY5 2017-01-11 2.0 Electron transfer complex of cytochrome c and cytochrome c oxidase at 2.0 angstrom resolution
1CRC 1996-03-08 2.08 CYTOCHROME C AT LOW IONIC STRENGTH
3NBT 2010-07-14 2.1 Crystal structure of trimeric cytochrome c from horse heart
4NFG 2014-09-24 2.11 K13R mutant of horse cytochrome c and yeast cytochrome c peroxidase complex
4RSZ 2015-01-14 2.19 The X-ray structure of the Primary Adduct formed in the Reaction between Cisplatin and Cytochrome c
3NBS 2010-07-14 2.2 Crystal structure of dimeric cytochrome c from horse heart
1KTD 2002-05-01 2.4 CRYSTAL STRUCTURE OF CLASS II MHC MOLECULE IEK BOUND TO PIGEON CYTOCHROME C PEPTIDE
1U75 2004-09-28 2.55 Electron Transfer Complex between Horse Heart Cytochrome c and Zinc-Porphyrin Substituted Cytochrome c Peroxidase
2PCB 1993-07-15 2.8 CRYSTAL STRUCTURE OF A COMPLEX BETWEEN ELECTRON TRANSFER PARTNERS, CYTOCHROME C PEROXIDASE AND CYTOCHROME C
3JBT 2015-11-18 3.8 Atomic structure of the Apaf-1 apoptosome
5JUY 2016-10-19 4.1 Active human apoptosome with procaspase-9
5WVE 2017-02-08 4.4 Apaf-1-Caspase-9 holoenzyme
3J2T 2013-04-10 9.5 An improved model of the human apoptosome

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.3 Cytochrome c P00021 CYC_COLLI
92.2 Cytochrome c P00020 CYC_ANAPL
92.3 Cytochrome c P81280 CYC_ALLMI
90.5 Cytochrome c Q52V10 CYC_SAISC
93.3 Cytochrome c P00012 CYC_MIRLE
93.3 Cytochrome c Q52V09 CYC_CEPBA
93.3 Cytochrome c P00013 CYC_MINSC
93.3 Cytochrome c P00014 CYC_MACGI
94.3 Cytochrome c P00011 CYC_CANLF
94.3 Cytochrome c P62898 CYC_RAT
94.3 Cytochrome c P00008 CYC_RABIT
94.3 Cytochrome c P62897 CYC_MOUSE
95.2 Cytochrome c P68098 CYC_LAMGU
95.2 Cytochrome c P68100 CYC_ESCRO
95.2 Cytochrome c P68099 CYC_CAMDR
94.3 Cytochrome c P00007 CYC_HIPAM
97.1 Cytochrome c P62896 CYC_SHEEP
97.1 Cytochrome c P62895 CYC_PIG
97.1 Cytochrome c P62894 CYC_BOVIN
99.0 Cytochrome c P68096 CYC_EQUBU
99.0 Cytochrome c P68097 CYC_EQUAS
100.0 Cytochrome c P00004 CYC_HORSE
90.3 Cytochrome c B4USV4 CYC_OTOGA