Kinetic mechanism of cytochrome c folding: involvement of the heme and its ligands.


Abstract

The covalently attached heme and its axial ligands not only are essential for the structure and function of cytochrome c but they also play an important role in the folding process. Under typical denaturing conditions (concentrated guanidine hydrochloride or urea near pH 7), one of the axial ligands, His 18, remains bound to the oxidized heme iron, but the second ligand, Met 80, is replaced by a non-native histidine ligand (His 26 or His 33 in horse cytochrome c). Using quenched-flow and NMR methods, hydrogen exchange rates were measured for several individual amide protons in guanidine-denatured horse cytochrome c. The observation of a single highly protected (140-fold) backbone amide, that of His 18, suggests the presence of a persistent H-bond consistent with heme ligation of the His 18 side chain in the unfolded state. Heme absorbance changes induced by rapid acidification of oxidized cytochrome c in 4.5 M guanidine hydrochloride from pH 7.8 to 4.6 or below exhibit two kinetic phases with rates of 110 and 25 s-1, attributed to the dissociation of non-native histidine ligands from the heme in the unfolded state. The kinetics of folding from guanidine-denatured cytochrome c under a variety of initial and final conditions was investigated by stopped-flow methods, using tryptophan fluorescence as a conformational probe and Soret absorbance as a probe for the ligation state of the heme. A fast kinetic phase (80 s-1) accompanied by a major decrease in fluorescence and a minor absorbance change coincides with the formation of a partially folded intermediate with interacting chain termini detected in earlier pulsed NH exchange measurements [Roder, H., Elöve, G. A., & Englander, S. W. (1988) Nature 335, 700]. At neutral pH, an intermediate kinetic phase (1.8 s-1) accounts for 78% of the absorbance change and 47% of the fluorescence change. In contrast, the folding kinetics at pH 5 is dominated by the fast phase, and the amplitude of the intermediate phase is reduced to approximately 10%. The pH-dependent amplitude changes show titration behavior with an apparent pK of approximately 5.7, consistent with the protonation of a single histidine residue. The intermediate phase can also be suppressed by the addition of 20 mM imidazole. Since both of these conditions interfere with histidine ligation, the intermediate kinetic phase is attributed to the presence of a non-native histidine ligand (His 26 or His 33) that can become trapped in a partially folded intermediate.(ABSTRACT TRUNCATED AT 400 WORDS) Study holds ProTherm entries: 4467, 4468 Extra Details:

Submission Details

ID: QyqozPLS3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:25 p.m.

Version: 1

Publication Details
Elöve GA;Bhuyan AK;Roder H,Biochemistry (1994) Kinetic mechanism of cytochrome c folding: involvement of the heme and its ligands. PMID:8204626
Additional Information

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
1GIW 1998-12-09 SOLUTION STRUCTURE OF REDUCED HORSE HEART CYTOCHROME C, NMR, MINIMIZED AVERAGE STRUCTURE
2FRC 1997-07-29 CYTOCHROME C (REDUCED) FROM EQUUS CABALLUS, NMR, MINIMIZED AVERAGE STRUCTURE
2N3B 2015-10-28 Structure of oxidized horse heart cytochrome c encapsulated in reverse micelles
1I5T 2001-03-21 SOLUTION STRUCTURE OF CYANOFERRICYTOCHROME C
1AKK 1997-09-17 SOLUTION STRUCTURE OF OXIDIZED HORSE HEART CYTOCHROME C, NMR, MINIMIZED AVERAGE STRUCTURE
1M60 2002-08-07 Solution Structure of Zinc-substituted cytochrome c
1FI7 2000-08-23 Solution structure of the imidazole complex of cytochrome C
2GIW 1998-12-09 SOLUTION STRUCTURE OF REDUCED HORSE HEART CYTOCHROME C, NMR, 40 STRUCTURES
1LC2 2003-06-03 Solution Structure Of Reduced Horse Heart Cytochrome c in 30% Acetonitrile Solution, NMR 30 Structures
1FI9 2000-08-23 SOLUTION STRUCTURE OF THE IMIDAZOLE COMPLEX OF CYTOCHROME C
1LC1 2003-06-03 Solution Structure Of Reduced Horse Heart Cytochrome c in 30% Acetonitrile Solution, NMR Minimized Average Structure
1OCD 1997-06-16 CYTOCHROME C (OXIDIZED) FROM EQUUS CABALLUS, NMR, MINIMIZED AVERAGE STRUCTURE
5ZKV 2019-05-22 Solution structure of molten globule state of L94G mutant of horse 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
1WEJ 1998-12-09 1.8 IGG1 FAB FRAGMENT (OF E8 ANTIBODY) COMPLEXED WITH HORSE CYTOCHROME C AT 1.8 A RESOLUTION
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
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