The effect of mutation at valine-45 on the stability and redox potentials of trypsin-cleaved cytochrome b5.


Abstract

In an attempt to elucidate the determinants of redox potential and protein stability in cytochrome b5, three mutants at a highly conserved residue Val45, which is a member of heme hydrophobic pocket residues have been characterized. The V45Y mutant was designed to introduce a bulkier residue and a hydroxyl group to the heme pocket. The mutants V45H and V45E were constructed to test the effect of positive and negative charge on the stability and redox potential of proteins. The influence of these mutants on the protein stability towards thermal, urea, acid, ethanol and on the redox potential were studied. It is concluded that the decrease of hydrophobic free energy and the larger volume of the tyrosine make the phenylhydroxyl group of tyrosine still sitting inside the hydrophobic pocket, while the side chain of the mutant V45E and V45H shift away from the heme pocket. The redox potentials of mutants V45Y, V45H, V45E and wild-type of cytochrome b5 are -35 mV, 8 mV, -26 mV and -3 mV, respectively. The bigger change of the V45Y on redox potential is due to the close contact between the hydroxyl group and the heme, while the changes of the V45E and V45H result from the alteration of charge density and distribution around the heme. Different relative stability of these mutants towards heat have been observed with the order: WT > V45Y-V45H > V45E being both in the oxidized and reduced state. The relative stability induced by addition of urea decreases in the order: WT > V45Y > V45H > V45E. These results suggest that the difference in the hydrophobic free energy is a major factor contributing to the stability of the Val45 mutants. Also the loose of the helix III in the mutant V45E makes it more unstable. These results indicate that residue Val45 plays an important role in the stability and redox potential of the protein. Study holds ProTherm entries: 9181, 9182, 9183, 9184, 9185, 9186, 9187, 9188, 9189, 9190, 9191, 9192, 14491, 14492, 14493, 14494, 14495, 14496 Extra Details: cytochrome b5; mutagenesis; redox potential; protein stability

Submission Details

ID: Ya4bbVSP3

Submitter: Connie Wang

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

Version: 1

Publication Details
Wang ZQ;Wang YH;Wang WH;Xue LL;Wu XZ;Xie Y;Huang ZX,Biophys. Chem. (2000) The effect of mutation at valine-45 on the stability and redox potentials of trypsin-cleaved cytochrome b5. PMID:10631476
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
1AQA 1997-09-17 SOLUTION STRUCTURE OF REDUCED MICROSOMAL RAT CYTOCHROME B5, NMR, MINIMIZED AVERAGE STRUCTURE
1JEX 2001-07-11 SOLUTION STRUCTURE OF A67V MUTANT OF RAT FERRO CYTOCHROME B5
1AXX 1998-03-04 THE SOLUTION STRUCTURE OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5, NMR, 19 STRUCTURES
1HKO 2003-03-18 NMR structure of bovine cytochrome b5
2M33 2013-02-06 Solution NMR structure of full-length oxidized microsomal rabbit cytochrome b5
1IB7 2001-04-04 SOLUTION STRUCTURE OF F35Y MUTANT OF RAT FERRO CYTOCHROME B5, A CONFORMATION, ENSEMBLE OF 20 STRUCTURES
1I8C 2001-05-16 SOLUTION STRUCTURE OF THE WATER-SOLUBLE FRAGMENT OF RAT HEPATIC APOCYTOCHROME B5
1F04 2000-06-21 SOLUTION STRUCTURE OF OXIDIZED BOVINE MICROSOMAL CYTOCHROME B5 MUTANT (E44A, E48A, E56A, D60A) AND ITS INTERACTION WITH CYTOCHROME C
1NX7 2004-06-15 Solution Structure of Oxidized Bovine Microsomal Cytochrome B5
1SH4 2004-08-10 Solution structure of oxidized bovine microsomal cytochrome B5 Mutant V45H
1I87 2001-05-16 SOLUTION STRUCTURE OF THE WATER-SOLUBLE FRAGMENT OF RAT HEPATIC APOCYTOCHROME B5
1BLV 1998-07-29 SOLUTION STRUCTURE OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5 IN THE PRESENCE OF 2 M GUANIDINIUM CHLORIDE: MONITORING THE EARLY STEPS IN PROTEIN UNFOLDING
1B5A 1998-06-17 RAT FERROCYTOCHROME B5 A CONFORMATION, NMR, 1 STRUCTURE
1DO9 2000-01-05 SOLUTION STRUCTURE OF OXIDIZED MICROSOMAL RABBIT CYTOCHROME B5. FACTORS DETERMINING THE HETEROGENEOUS BINDING OF THE HEME.
5XEE 2018-02-14 NMR solution structure of the aromatic mutant H43F H67F cytochrome b5
5XE4 2018-02-14 NMR solution structure of the aromatic mutant H43W H67F cytochrome b5
1B5B 1998-06-17 RAT FERROCYTOCHROME B5 B CONFORMATION, NMR, 1 STRUCTURE
2I96 2007-09-04 Solution structure of the oxidized microsomal human cytochrome b5
1MNY 2002-11-13 Dimethyl propionate ester heme-containing cytochrome b5
1IEU 1997-04-21 APOCYTOCHROME B5, PH 6.2, 298 K, NMR, 10 STRUCTURES
1AW3 1998-02-04 THE SOLUTION NMR STRUCTURE OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5, MINIMIZED AVERAGE STRUCTURE
1I5U 2001-03-21 SOLUTION STRUCTURE OF CYTOCHROME B5 TRIPLE MUTANT (E48A/E56A/D60A)
1J0Q 2003-08-12 Solution Structure of Oxidized Bovine Microsomal Cytochrome b5 mutant V61H
1BFX 1998-08-12 THE SOLUTION NMR STRUCTURE OF THE B FORM OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5, MINIMIZED AVERAGE STRUCTURE
1F03 2000-06-21 SOLUTION STRUCTURE OF OXIDIZED BOVINE MICROSOMAL CYTOCHROME B5 MUTANT (E44A, E48A, E56A, D60A) AND ITS INTERACTION WITH CYTOCHROME C
2AXX 1998-03-04 THE SOLUTION STRUCTURE OF OXIDIZED RAT MICROSOMAL CYTOCHROME B5, NMR, 21 STRUCTURES
1IET 1997-04-21 APOCYTOCHROME B5, PH 6.2, 298 K, NMR, MINIMIZED AVERAGE STRUCTURE
3X34 2015-07-15 0.76 Crystal structure of the reduced form of the solubilized domain of porcine cytochrome b5 in form 1 crystal
3X32 2015-07-15 0.83 Crystal structure of the oxidized form of the solubilized domain of porcine cytochrome b5 in form 1 crystal
3X33 2015-07-15 0.93 Crystal structure of the oxidized form of the solubilized domain of porcine cytochrome b5 in form 2 crystal
3X35 2015-07-15 0.95 Crystal structure of the reduced form of the solubilized domain of porcine cytochrome b5 in form 2 crystal
1CYO 1994-11-30 1.5 BOVINE CYTOCHROME B(5)
3OZZ 2011-09-14 1.7 Structure of a cytochrome b5 core-swap mutant
1M20 2002-09-11 1.8 Crystal Structure of F35Y Mutant of Trypsin-solubilized Fragment of Cytochrome b5
1M2M 2003-03-18 1.8 Crystal structure of E44A/E48A/E56A/D60A mutant of cytochrome b5
1M2I 2003-03-18 1.8 Crystal structure of E44A/E56A mutant of cytochrome b5
1LQX 2002-09-04 1.8 Crystal structure of V45E mutant of cytochrome b5
1U9U 2005-02-01 1.86 Crystal structure of F58Y mutant of cytochrome b5
1LR6 2002-09-04 1.9 Crystal structure of V45Y mutant of cytochrome b5
1EHB 2000-08-09 1.9 CRYSTAL STRUCTURE OF RECOMBINANT TRYPSIN-SOLUBILIZED FRAGMENT OF CYTOCHROME B5
1M59 2003-03-18 1.9 Crystal Structure of P40V Mutant of Trypsin-solubilized Fragment of Cytochrome b5
2YBB 2011-10-19 19.0 Fitted model for bovine mitochondrial supercomplex I1III2IV1 by single particle cryo-EM (EMD-1876)
1U9M 2005-02-01 2.0 Crystal structure of F58W mutant of cytochrome b5
1ES1 2000-08-09 2.1 CRYSTAL STRUCTURE OF VAL61HIS MUTANT OF TRYPSIN-SOLUBILIZED FRAGMENT OF CYTOCHROME B5
1PP9 2004-07-20 2.1 Bovine cytochrome bc1 complex with stigmatellin bound
1PPJ 2004-07-20 2.1 Bovine cytochrome bc1 complex with stigmatellin and antimycin
2A06 2005-06-21 2.1 Bovine cytochrome bc1 complex with stigmatellin bound
2FYU 2006-08-29 2.26 Crystal structure of bovine heart mitochondrial bc1 with jg144 inhibitor
1L0L 2003-04-08 2.35 structure of bovine mitochondrial cytochrome bc1 complex with a bound fungicide famoxadone
4HIN 2013-10-16 2.4 2.4A Resolution Structure of Bovine Cytochrome b5 (S71L)
1NTM 2003-10-07 2.4 Crystal Structure of Mitochondrial Cytochrome bc1 Complex at 2.4 Angstrom
1NTZ 2003-10-07 2.6 Crystal Structure of Mitochondrial Cytochrome bc1 Complex Bound with Ubiquinone
1SQX 2005-09-06 2.6 Crystal Structure Analysis of Bovine Bc1 with Stigmatellin A
1L0N 2003-04-08 2.6 native structure of bovine mitochondrial cytochrome bc1 complex
1NTK 2003-10-07 2.6 Crystal Structure of Mitochondrial Cytochrome bc1 in Complex with Antimycin A1
5KLV 2016-10-12 2.65 Structure of bos taurus cytochrome bc1 with fenamidone inhibited
1SQB 2004-09-07 2.69 Crystal Structure Analysis of Bovine Bc1 with Azoxystrobin
1SQP 2005-11-01 2.7 Crystal Structure Analysis of Bovine Bc1 with Myxothiazol
1QCR 1998-10-14 2.7 CRYSTAL STRUCTURE OF BOVINE MITOCHONDRIAL CYTOCHROME BC1 COMPLEX, ALPHA CARBON ATOMS ONLY
6NHG 2019-06-19 2.8 Rhodobacter sphaeroides Mitochondrial respiratory chain complex
1SQV 2005-09-06 2.85 Crystal Structure Analysis of Bovine Bc1 with UHDBT
1SQQ 2005-10-25 3.0 Crystal Structure Analysis of Bovine Bc1 with Methoxy Acrylate Stilbene (MOAS)
1BE3 1999-01-13 3.0 CYTOCHROME BC1 COMPLEX FROM BOVINE
1BGY 1999-01-06 3.0 CYTOCHROME BC1 COMPLEX FROM BOVINE
5OKD 2018-02-28 3.1 Crystal structure of bovine Cytochrome bc1 in complex with inhibitor SCR0911.
1NU1 2003-10-07 3.2 Crystal Structure of Mitochondrial Cytochrome bc1 Complexed with 2-nonyl-4-hydroxyquinoline N-oxide (NQNO)
6HAW 2019-01-16 3.45 Crystal structure of bovine cytochrome bc1 in complex with 2-pyrazolyl quinolone inhibitor WDH2G7
5NMI 2017-06-14 3.5 Cytochrome bc1 bound to the inhibitor MJM170
4D6T 2015-01-14 3.57 Cytochrome bc1 bound to the 4(1H)-pyridone GW844520
4D6U 2015-01-14 4.09 Cytochrome bc1 bound to the 4(1H)-pyridone GSK932121
6FO0 2018-02-28 4.1 CryoEM structure of bovine cytochrome bc1 in complex with the anti-malarial compound GSK932121
6FO6 2018-02-28 4.1 CryoEM structure of bovine cytochrome bc1 in complex with the anti-malarial inhibitor SCR0911
6FO2 2018-02-28 4.4 CryoEM structure of bovine cytochrome bc1 with no ligand bound
5GPN 2017-04-05 5.4 Architecture of mammalian respirasome
5LUF 2016-11-30 9.1 Cryo-EM of bovine respirasome

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.3 Cytochrome b Q36291 CYB_MORKA
90.2 Cytochrome b O20965 CYB_TRAIM
90.2 Cytochrome b Q955G5 CYB_LITWA
90.2 Cytochrome b Q5UVI3 CYB_RUCDU
90.2 Cytochrome b Q8HEM1 CYB_RUCEL
91.0 Cytochrome b O47714 CYB_PELCP
91.0 Cytochrome b Q1H8Y8 CYB_MEGGI
90.2 Cytochrome b Q36324 CYB_BABBA
90.8 Cytochrome b Q6V8Y0 CYB_CEREN
91.0 Cytochrome b Q6V8Y1 CYB_CEREO
90.5 Cytochrome b Q5UVI6 CYB_BLADC
90.5 Cytochrome b Q4VKI6 CYB_HEMHY
90.2 Cytochrome b O78787 CYB_CAPCY
91.0 Cytochrome b P24957 CYB_GIRCA
91.0 Cytochrome b Q9T5N4 CYB_SAITA
90.8 Cytochrome b O47930 CYB_HYDIN
91.3 Cytochrome b Q9TGH8 CYB_RAPME
91.0 Cytochrome b O99345 CYB_MADKI
92.1 Cytochrome b Q9TDQ5 CYB_MUNVU
91.0 Cytochrome b O47721 CYB_MADGU
91.3 Cytochrome b O47924 CYB_CEREL
90.2 Cytochrome b O78781 CYB_AMMLE
91.8 Cytochrome b Q75R30 CYB_CERNY
91.8 Cytochrome b Q34172 CYB_CERNI
91.6 Cytochrome b Q9T9B7 CYB_TRASR
90.2 Cytochrome b P24964 CYB_PIG
91.6 Cytochrome b O99342 CYB_KOBEL
90.2 Cytochrome b Q33500 CYB_HIPAM
90.8 Cytochrome b Q5BQG9 CYB_SUSPH
91.0 Cytochrome b O03363 CYB_HEXLI
90.8 Cytochrome b Q35130 CYB_NAECA
90.2 Cytochrome b P38594 CYB_LEPWE
91.8 Cytochrome b P24955 CYB_DAMDA
91.6 Cytochrome b Q85RV3 CYB_MUNCR
90.8 Cytochrome b Q85IN4 CYB_MELMS
91.6 Cytochrome b O47923 CYB_CAPCA
91.0 Cytochrome b Q34891 CYB_LAMGL
91.3 Cytochrome b O47716 CYB_DAMLU
91.3 Cytochrome b O78785 CYB_CAPIB
91.6 Cytochrome b Q8M708 CYB_PHAAE
91.6 Cytochrome b Q9T9A8 CYB_ORYLE
91.8 Cytochrome b Q5UVJ0 CYB_DAMME
91.8 Cytochrome b Q9XP68 CYB_PSENG
91.0 Cytochrome b Q8M706 CYB_PHAAF
91.6 Cytochrome b Q5UVI5 CYB_PUDPU
91.0 Cytochrome b Q9MMZ2 CYB_OREAM
91.8 Cytochrome b O99338 CYB_RAPSH
92.9 Cytochrome b P24960 CYB_ODOHE
92.1 Cytochrome b O79327 CYB_CEPLE
91.3 Cytochrome b Q9ZZR4 CYB_OVIMU
91.3 Cytochrome b O78783 CYB_HEMJE
92.6 Cytochrome b Q94QD3 CYB_ELADA
91.8 Cytochrome b O47723 CYB_ANTMR
92.1 Cytochrome b O47718 CYB_RAPCA
91.3 Cytochrome b O78778 CYB_OVIAD
91.3 Cytochrome b Q4VKI7 CYB_ARAJA
92.1 Cytochrome b O99257 CYB_ALCLI
91.3 Cytochrome b O78779 CYB_OVIDA
91.8 Cytochrome b Q9B5Q8 CYB_CEPSI
92.3 Cytochrome b Q9TGH6 CYB_ANTCE
92.1 Cytochrome b O78773 CYB_ADDNA
92.6 Cytochrome b Q9T9B6 CYB_TRAOR
92.3 Cytochrome b Q36058 CYB_TRAJA
92.3 Cytochrome b Q34760 CYB_HIPNI
92.9 Cytochrome b P92592 CYB_BUBQU
92.9 Cytochrome b Q34717 CYB_HIPEQ
91.8 Cytochrome b Q9B5S0 CYB_CEPDO
93.1 Cytochrome b O20964 CYB_TRAEU
91.6 Cytochrome b O78775 CYB_PANHO
91.8 Cytochrome b Q9B5Q9 CYB_CEPOG
92.3 Cytochrome b O47926 CYB_CAPPY
91.6 Cytochrome b O78789 CYB_CAPHE
92.6 Cytochrome b O47922 CYB_ALCAA
92.1 Cytochrome b O78776 CYB_RUPRU
92.9 Cytochrome b O79360 CYB_KOBME
92.9 Cytochrome b Q7J370 CYB_KOBVA
92.9 Cytochrome b O78858 CYB_KOBKO
92.3 Cytochrome b Q9B5Q1 CYB_SYLGR
92.1 Cytochrome b O78786 CYB_CAPFA
92.1 Cytochrome b O78782 CYB_PSENA
92.9 Cytochrome b O20968 CYB_TRASP
93.1 Cytochrome b Q9TDQ7 CYB_MUNMU
92.1 Cytochrome b Q9B5Q5 CYB_CEPRF
92.3 Cytochrome b O78777 CYB_RUPPY
92.3 Cytochrome b Q9B5Q7 CYB_CEPSP
92.9 Cytochrome b Q7HEI2 CYB_ALCAC
92.1 Cytochrome b P24965 CYB_TRANA
92.6 Cytochrome b P24992 CYB_ANTAM
92.9 Cytochrome b O48336 CYB_GAZGA
92.3 Cytochrome b Q9B1D2 CYB_CEPNA
92.6 Cytochrome b Q9B5R7 CYB_CEPJE
92.3 Cytochrome b Q7IYN8 CYB_CEPHR
92.9 Cytochrome b Q9XLE0 CYB_REDRE
93.1 Cytochrome b Q8M0K9 CYB_MUNRE
92.1 Cytochrome b Q9B5Q6 CYB_CEPRU
92.3 Cytochrome b Q9B5R1 CYB_CEPNI
91.8 Cytochrome b O78784 CYB_CAPSI
92.1 Cytochrome b P24953 CYB_CAPHI
93.4 Cytochrome b O99343 CYB_TAUDE
92.6 Cytochrome b Q9B5Q3 CYB_CEPZE
92.3 Cytochrome b Q9B5R0 CYB_CEPNG
92.9 Cytochrome b Q9XLE2 CYB_REDAR
92.6 Cytochrome b Q9B5R8 CYB_CEPCA
92.6 Cytochrome b Q9B5S1 CYB_CEPAD
93.1 Cytochrome b O47583 CYB_MOSMO
92.9 Cytochrome b O48309 CYB_MOSFU
92.9 Cytochrome b Q7J6I1 CYB_MOSCH
93.9 Cytochrome b Q33950 CYB_BUBBU
92.9 Cytochrome b Q9G3T7 CYB_OUROU
93.1 Cytochrome b O47584 CYB_MOSLE
93.9 Cytochrome b P92870 CYB_BUBDE
92.6 Cytochrome b Q9B5R3 CYB_PHIMO
93.1 Cytochrome b Q9T9A6 CYB_OREOR
93.4 Cytochrome b Q9T9B2 CYB_GAZSU
93.7 Cytochrome b O78774 CYB_NANGR
92.9 Cytochrome b Q9B5R2 CYB_PHIMA
92.9 Cytochrome b Q35273 CYB_OVIMO
92.9 Cytochrome b Q9B5Q4 CYB_CEPWE
93.1 Cytochrome b Q34045 CYB_CAPCR
93.7 Cytochrome b Q9TG16 CYB_TRAAN
93.4 Cytochrome b O47420 CYB_BEAHU
93.7 Cytochrome b O79426 CYB_NEOMO
93.4 Cytochrome b O79440 CYB_ORYDA
93.4 Cytochrome b O78742 CYB_ALCBU
93.7 Cytochrome b Q955G6 CYB_ORYGA
93.9 Cytochrome b Q9T9A7 CYB_DAMPY
93.9 Cytochrome b O79311 CYB_BOSTR
93.4 Cytochrome b O99258 CYB_CONGN
94.2 Cytochrome b P92584 CYB_BUBMI
93.9 Cytochrome b O47421 CYB_CONTA
94.5 Cytochrome b Q9T9B9 CYB_SYNCA
94.2 Cytochrome b Q9XLE1 CYB_REDFU
93.9 Cytochrome b Q5C9H5 CYB_CAPSU
94.7 Cytochrome b Q9T9C0 CYB_TETQU
96.3 Cytochrome b Q9T9C1 CYB_BISBI
97.1 Cytochrome b O20998 CYB_BISBO
97.1 Cytochrome b Q5Y4Q0 CYB_BOSMU
96.8 Cytochrome b Q5QD74 CYB_BOSSA
97.6 Cytochrome b Q5QD76 CYB_BOSGF
97.9 Cytochrome b P92583 CYB_BOSJA
99.5 Cytochrome b Q8HCJ4 CYB_BOSIN
100.0 Cytochrome b P00157 CYB_BOVIN
92.9 Cytochrome b5 P00168 CYB5_ALOSE
90.2 Cytochrome b5 P56395 CYB5_MOUSE
91.3 Cytochrome b5 P00173 CYB5_RAT
90.2 Cytochrome b5 P00167 CYB5_HUMAN
91.3 Cytochrome b5 P00169 CYB5_RABIT
91.3 Cytochrome b5 P00170 CYB5_HORSE
96.7 Cytochrome b5 P00172 CYB5_PIG
100.0 Cytochrome b5 P00171 CYB5_BOVIN