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


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 UniProtKB Entries

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