Kinetic barriers to the folding of horse cytochrome C in the reduced state.


Abstract

To determine the kinetic barrier in the folding of horse cytochrome c, a CO-liganded derivative of cytochrome c, called carbonmonoxycytochrome c, has been prepared by exploiting the thermodynamic reversibility of ferrocytochrome c unfolding induced by guanidinium hydrochloride (GdnHCl), pH 7. The CO binding properties of unfolded ferrocytochrome c, studied by 13C NMR and optical spectroscopy, are remarkably similar to those of native myoglobin and isolated chains of human hemoglobin. Equilibrium unfolding transitions of ferrocytochrome c in the presence and the absence of CO observed by both excitation energy transfer from the lone tryptophan to the ferrous heme and far-UV circular dichroism (CD) indicate no accumulation of structural intermediates to a detectable level. Values of thermodynamic parameters obtained by two-state analysis of fluorescence transitions are DeltaG(H2O) = 11.65(+/-1.13) kcal x mol(-1) and C(m) = 3.9(+/-0.1) M GdnHCl in the presence of CO, and DeltaG(H2O)=19.3(+/-0.5) kcal x mol(-1) and C(m) = 5.1(+/-0.1) M GdnHCl in the absence of CO, indicating destabilization of ferrocytochrome c by approximately 7.65 kcal x mol(-1) due to CO binding. The native states of ferrocytochrome c and carbonmonoxycytochrome c are nearly identical in terms of structure and conformation except for the Fe2+-M80 --> Fe2+-CO replacement. Folding and unfolding kinetics as a function of GdnHCl, studied by stopped-flow fluorescence, are significantly different for the two proteins. Both refold fast, but carbonmonoxycytochrome c refolds 2-fold faster (tau = 1092 micros at 10 degrees C) than ferrocytochrome c. Linear extrapolation of the folding rates to the ordinate of the chevron plot projects this value of tau to 407 micros. The unfolding rate of the former in water, estimated by extrapolation, is faster by more than 10 orders of magnitude. Significant differences are also observed in rate-denaturant gradients in the chevron. Formation and disruption of the Fe2+-M80 coordination contact clearly impose high-energy kinetic barriers to folding and unfolding of ferrocytochrome c. The unfolding barrier due to the Fe2+-M80 bond provides sufficient kinetic stability to the native state of ferrocytochrome c to perform its physiological function as an electron donor. Study holds ProTherm entries: 15654, 15655, 15656, 15657 Extra Details: kinetic barrier; thermodynamic reversibility; folding rate; electron donor

Submission Details

ID: uUd8fZSv

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:46 p.m.

Version: 1

Publication Details
Bhuyan AK;Kumar R,Biochemistry (2002) Kinetic barriers to the folding of horse cytochrome C in the reduced state. PMID:12379125
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
1KTD 2002-01-15T00:00:00+0000 2.4 CRYSTAL STRUCTURE OF CLASS II MHC MOLECULE IEK BOUND TO PIGEON CYTOCHROME C PEPTIDE
2B4Z 2005-09-27T00:00:00+0000 1.5 Crystal structure of cytochrome C from bovine heart at 1.5 A resolution.
2YBB 2011-03-02T00:00:00+0000 19.0 Fitted model for bovine mitochondrial supercomplex I1III2IV1 by single particle cryo-EM (EMD-1876)
3J2T 2012-12-23T00:00:00+0000 9.5 An improved model of the human apoptosome
5C0Z 2015-06-12T00:00:00+0000 1.12 The structure of oxidized rat cytochrome c at 1.13 angstroms resolution
5C9M 2015-06-28T00:00:00+0000 1.36 The structure of oxidized rat cytochrome c (T28A) at 1.362 angstroms resolution.
5DF5 2015-08-26T00:00:00+0000 1.3 The structure of oxidized rat cytochrome c (T28E) at 1.30 angstroms resolution.
5JUY 2016-05-10T00:00:00+0000 4.1 Active human apoptosome with procaspase-9
6FF5 2018-01-03T00:00:00+0000 1.74 X-ray structure of bovine heart cytochrome c at high ionic strength
6N1O 2018-11-09T00:00:00+0000 1.55 Oxidized rat cytochrome c mutant (S47E)

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