Mutations of iso-1-cytochrome c at positions 13 and 90. Separate effects on physical and functional properties.


Abstract

Residues at positions 13 (lysine or arginine) and 90 (glutamate or aspartate) of eukaryotic cytochromes c have been conserved during evolution; Cys102, however, is found only in yeast cytochrome c. The positively charged residue at position 13 and the negatively charged residue at position 90 are close together in those cytochromes c for which three-dimensional structures are available. We have replaced the amino acids at these two positions by cysteine in Saccharomyces cerevisiae iso-1-cytochrome c; in an earlier study, Cys102 was replaced by threonine without negatively influencing the physical or enzymic properties of the protein. The mutated proteins [R13C, C102T]cytochrome c (iso-1-cytochrome c containing Arg13-->Cys and Cys102-->Thr mutations), [D90C, C102T]cytochrome c (iso-1-cytochrome c containing Asp90-->Cys and Cys102-->Thr mutations) and [R13C, D90C, C102T]cytochrome c (iso-1-cytochrome c containing Arg13-->Cys, Asp90-->Cys, and Cys102-->Thr mutations) are functional in vivo. Free sulfhydryl titration shows that the doubly mutated forms each contain one sulfhydryl group while the triple mutant contains two sulfhydryl groups. The stability of mutant [R13C, C102T]cytochrome c resembles that of [C102T] cytochrome c, whereas the stability of [D90C, C102T]cytochrome c resembles the stability of [R13C, D90C, C102T]cytochrome c. The activity of cytochrome-c oxidase using cytochrome c was monitored polarographically. Compared to the wild-type or [C102T]cytochrome c, which shows two kinetic phases with cytochrome-c oxidase, [D90C, C102T]cytochrome c has much the same profile; [R13C, C102T]cytochrome c and [R13C, D90C, C102T]cytochrome c exhibit one kinetic phase with decreased activity. Electron-transfer activity of the mutant cytochromes c is inhibited by Hg2+. The inhibition is highest for the triple mutant, less for [R13C, C102T]cytochrome c, even less for [D90C, C102T]cytochrome c and insignificant for the wild type. It would appear as though the stability of the triple mutant follows the changes that result from the Asp90-->Cys mutation while the activity changes follow those of the Arg13-->Cys mutation. Study holds ProTherm entries: 7166, 7167, 7168, 7169, 7170 Extra Details: cysteine; sulfhydryl group; polarographic;,electron-transfer activity

Submission Details

ID: fVdJJ4iC4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:33 p.m.

Version: 1

Publication Details
Huang Y;Beeser S;Guillemette JG;Storms RK;Kornblatt JA,Eur. J. Biochem. (1994) Mutations of iso-1-cytochrome c at positions 13 and 90. Separate effects on physical and functional properties. PMID:8033888
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
1NMI 2003-02-04 Solution structure of the imidazole complex of iso-1 cytochrome c
2GB8 2006-11-21 Solution structure of the complex between yeast iso-1-cytochrome c and yeast cytochrome c peroxidase
2ORL 2007-04-24 Solution structure of the cytochrome c- para-aminophenol adduct
1FHB 1995-09-15 THREE-DIMENSIONAL SOLUTION STRUCTURE OF THE CYANIDE ADDUCT OF A MET80ALA VARIANT OF SACCHAROMYCES CEREVISIAE ISO-1-CYTOCHROME C. IDENTIFICATION OF LIGAND-RESIDUE INTERACTIONS IN THE DISTAL HEME CAVITY
2N18 2015-05-13 Dominant form of the low-affinity complex of yeast cytochrome c and cytochrome c peroxidase
2LIT 2011-12-07 NMR Solution Structure of Yeast Iso-1-cytochrome c Mutant P71H in reduced states
1LMS 2003-03-18 Structural model for an alkaline form of ferricytochrome c
1YFC 1997-03-12 Solution nmr structure of a yeast iso-1-ferrocytochrome C
2JQR 2008-04-22 Solution model of crosslinked complex of cytochrome c and adrenodoxin
2HV4 2006-09-26 NMR solution structure refinement of yeast iso-1-ferrocytochrome c
2JTI 2008-07-22 Solution structure of the yeast iso-1-cytochrome c (T12A) : yeast cytochrome c peroxidase complex
2MHM 2014-10-29 Solution structure of cytochrome c Y67H
2LIR 2011-12-07 NMR Solution Structure of Yeast Iso-1-cytochrome c Mutant P71H in oxidized states
1YIC 1997-07-23 THE OXIDIZED SACCHAROMYCES CEREVISIAE ISO-1-CYTOCHROME C, NMR, 20 STRUCTURES
4N0K 2014-09-10 1.05 Atomic resolution crystal structure of a cytochrome c-calixarene complex
6GD6 2018-08-29 1.2 Cytochrome c in complex with Sulfonato-calix[8]arene, H3 form with ammonium sulfate
1YCC 1991-07-15 1.23 HIGH-RESOLUTION REFINEMENT OF YEAST ISO-1-CYTOCHROME C AND COMPARISONS WITH OTHER EUKARYOTIC CYTOCHROMES C
5LFT 2017-07-12 1.25 Crystal structure of cytochrome c - Bromo-trisulfonatocalix[4]arene complexes
4YE1 2015-05-13 1.39 A cytochrome c plus calixarene structure - alternative ligand binding mode
3TYI 2012-05-02 1.4 Crystal Structure of Cytochrome c - p-Sulfonatocalix[4]arene Complexes
4MU8 2014-06-04 1.45 Crystal structure of an oxidized form of yeast iso-1-cytochrome c at pH 8.8
5NCV 2018-03-14 1.5 Crystal Structure of Cytochrome c in complex with p-Methylphosphonatocalix[4]arene
6GD7 2018-08-29 1.55 Cytochrome c in complex with Sulfonato-calix[8]arene, H3 form with PEG
5T8W 2017-11-01 1.6 Cytochrome c - calixarene free
5KKE 2017-03-22 1.7 Crystal Structure of a Domain-swapped Dimer of Yeast Iso-1-cytochrome c with CYMAL5
5KPF 2017-07-12 1.7 Crystal structure of cytochrome c - Phenyl-trisulfonatocalix[4]arene complex
2BCN 2006-02-28 1.7 Solvent isotope effects on interfacial protein electron transfer between cytochrome c and cytochrome c peroxidase
1CIE 1995-01-26 1.8 STRUCTURAL AND FUNCTIONAL EFFECTS OF MULTIPLE MUTATIONS AT DISTAL SITES IN CYTOCHROME C
1CIG 1995-01-26 1.8 STRUCTURAL AND FUNCTIONAL EFFECTS OF MULTIPLE MUTATIONS AT DISTAL SITES IN CYTOCHROME C
1CIH 1995-01-26 1.8 STRUCTURAL AND FUNCTIONAL EFFECTS OF MULTIPLE MUTATIONS AT DISTAL SITES IN CYTOCHROME C
5LYC 2017-05-10 1.8 Cytochrome c in complex with phosphonato-calix[6]arene
1CSU 1995-01-26 1.81 REPLACEMENTS IN A CONSERVED LEUCINE CLUSTER IN THE HYDROPHOBIC HEME POCKET OF CYTOCHROME C
4Q5P 2015-04-22 1.87 Lysine-Ligated Yeast Iso-1 Cytochrome C
1S6V 2004-04-27 1.88 Structure of a cytochrome c peroxidase-cytochrome c site specific cross-link
1CRH 1994-01-31 1.9 THE ROLE OF A CONSERVED INTERNAL WATER MOLECULE AND ITS ASSOCIATED HYDROGEN BOND NETWORK IN CYTOCHROME C
1CSX 1995-01-26 1.9 REPLACEMENTS IN A CONSERVED LEUCINE CLUSTER IN THE HYDROPHOBIC HEME POCKET OF CYTOCHROME C
1CSW 1995-01-26 1.9 REPLACEMENTS IN A CONSERVED LEUCINE CLUSTER IN THE HYDROPHOBIC HEME POCKET OF CYTOCHROME C
1CSV 1995-01-26 1.9 REPLACEMENTS IN A CONSERVED LEUCINE CLUSTER IN THE HYDROPHOBIC HEME POCKET OF CYTOCHROME C
1CHJ 1994-12-20 1.9 STRUCTURAL STUDIES OF THE ROLES OF RESIDUES 82 AND 85 AT THE INTERACTIVE FACE OF CYTOCHROME C
1CTZ 1993-07-15 1.9 MUTATION OF TYROSINE-67 IN CYTOCHROME C SIGNIFICANTLY ALTERS THE LOCAL HEME ENVIRONMENT
2YCC 1992-07-15 1.9 OXIDATION STATE-DEPENDENT CONFORMATIONAL CHANGES IN CYTOCHROME C
1IRV 1997-01-11 1.9 CYTOCHROME C ISOZYME 1, REDUCED, MUTANT WITH ILE 75 REPLACED BY MET AND CYS 102 REPLACED BY THR
3CX5 2008-05-13 1.9 Structure of complex III with bound cytochrome c in reduced state and definition of a minimal core interface for electron transfer.
1CIF 1995-01-26 1.9 STRUCTURAL AND FUNCTIONAL EFFECTS OF MULTIPLE MUTATIONS AT DISTAL SITES IN CYTOCHROME C
1RAQ 1993-10-31 1.9 THE STRUCTURE AND FUNCTION OF OMEGA LOOP A REPLACEMENTS IN CYTOCHROME C
1CHH 1994-12-20 1.97 STRUCTURAL STUDIES OF THE ROLES OF RESIDUES 82 AND 85 AT THE INTERACTIVE FACE OF CYTOCHROME C
5KLU 2017-03-22 1.99 Crystal Structure of a Domain-swapped Dimer of Yeast Iso-1-cytochrome c with omega-undecylenyl-beta-D-maltopyranoside
1CRG 1994-01-31 2.0 THE ROLE OF A CONSERVED INTERNAL WATER MOLECULE AND ITS ASSOCIATED HYDROGEN BOND NETWORK IN CYTOCHROME C
1IRW 1997-01-11 2.0 CYTOCHROME C ISOZYME 1, REDUCED, MUTANT WITH ASN 52 REPLACED BY ALA AND CYS 102 REPLACED BY THR
1CHI 1994-12-20 2.0 STRUCTURAL STUDIES OF THE ROLES OF RESIDUES 82 AND 85 AT THE INTERACTIVE FACE OF CYTOCHROME C
5T7H 2017-03-22 2.0 Crystal structure of dimeric yeast iso-1-cytochrome C with CYMAL6
1CRI 1994-01-31 2.0 THE ROLE OF A CONSERVED INTERNAL WATER MOLECULE AND ITS ASSOCIATED HYDROGEN BOND NETWORK IN CYTOCHROME C
5CIF 2016-08-03 2.01 Complex of yeast cytochrome c peroxidase (W191F) with iso-1 cytochrome c
4P4Q 2015-03-25 2.01 Complex of yeast cytochrome c peroxidase (W191F) with iso-1 cytochrome c
1CRJ 1994-01-31 2.05 THE ROLE OF A CONSERVED INTERNAL WATER MOLECULE AND ITS ASSOCIATED HYDROGEN BOND NETWORK IN CYTOCHROME C
5CIG 2016-08-03 2.06 Complex of yeast cytochrome c peroxidase (W191G) with iso-1 cytochrome c
5CIC 2016-08-03 2.1 Complex of yeast cytochrome c peroxidase (W191G) bound to 3-aminobenzotrifluoride with iso-1 cytochrome c
4QAO 2015-08-05 2.1 Lysine-ligated cytochrome c with F82H
6EGZ 2018-10-10 2.17 Crystal structure of cytochrome c in complex with di-PEGylated sulfonatocalix[4]arene
1CTY 1993-07-15 2.2 MUTATION OF TYROSINE-67 IN CYTOCHROME C SIGNIFICANTLY ALTERS THE LOCAL HEME ENVIRONMENT
1RAP 1993-10-31 2.25 THE STRUCTURE AND FUNCTION OF OMEGA LOOP A REPLACEMENTS IN CYTOCHROME C
2B11 2005-10-25 2.3 Crystal structure of the protein-protein complex between F82W cytochrome c and cytochrome c peroxidase
2PCC 1993-07-15 2.3 CRYSTAL STRUCTURE OF A COMPLEX BETWEEN ELECTRON TRANSFER PARTNERS, CYTOCHROME C PEROXIDASE AND CYTOCHROME C
1U74 2004-09-28 2.4 Electron Transfer Complex between cytochrome C and cytochrome C peroxidase
6GD8 2018-08-29 2.5 Cytochrome c in complex with Sulfonato-calix[8]arene, P31 form
5CIH 2016-08-03 2.6 Complex of yeast cytochrome c peroxidase (W191Y) with iso-1 cytochrome c
5CIE 2016-08-03 2.6 Complex of yeast cytochrome c peroxidase (W191G) bound to aniline with iso-1 cytochrome c
6RGI 2019-07-10 2.64 Partially unfolded cytochrome c in complex with sulfonatocalix[6]arene
6GD9 2018-08-29 2.65 Cytochrome c in complex with Sulfonato-calix[8]arene, P43212 form
6EGY 2018-10-10 2.7 Crystal structure of cytochrome c in complex with mono-PEGylated sulfonatocalix[4]arene
2B0Z 2005-10-25 2.7 Crystal structure of the protein-protein complex between F82I cytochrome c and cytochrome c peroxidase
5CID 2016-08-03 2.76 Complex of yeast cytochrome c peroxidase (W191G) bound to o-toluidine with iso-1 cytochrome c
2B10 2005-10-25 2.8 Crystal Structure of the Protein-Protein Complex between F82S cytochrome c and cytochrome c peroxidase
6GDA 2019-05-01 2.8 Cytochrome c in complex with Sulfonato-calix[8]arene, P43212 form soaked with Spermine
1KYO 2002-03-06 2.97 YEAST CYTOCHROME BC1 COMPLEX WITH BOUND SUBSTRATE CYTOCHROME C
5CIB 2016-08-03 3.01 Complex of yeast cytochrome c peroxidase (W191G) bound to 2,4-dimethylaniline with iso-1 cytochrome c
2B12 2005-10-25 3.02 Crystal structure of the protein-protein complex between F82Y cytochrome c and cytochrome c peroxidase

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.0 Cytochrome c iso-1 P25400 CYC_CANGA
100.0 Cytochrome c iso-1 P00044 CYC1_YEAST