Effect of mutation at valine 61 on the three-dimensional structure, stability, and redox potential of cytochrome b5.


To elucidate the role played by Val61 of cytochrome b(5), this residue of the tryptic fragment of bovine liver cytochrome b(5) was chosen for replacement with tyrosine (Val61Tyr), histidine (Val61His), glutamic acid (Val61Glu), and lysine (Val61Lys) by means of site-directed mutagenesis. The mutants Val61Tyr, Val61Glu, Val61His, and Val61Lys exhibit electronic spectra identical to that of the wild type, suggesting that mutation at Val61 did not affect the overall protein structure significantly. The redox potentials determined by differential pulse voltammetry were -10 (wild type), -25 (Val61Glu), -33 (Val61Tyr), 12 (Val61His), and 17 mV (Val61Lys) versus NHE. The thermal stabilities and urea-mediated denaturation of wild-type cytochrome b(5) and its mutants were in the following order: wild type > Val61Glu > Val61Tyr > Val61His > Val61Lys. The kinetics of denaturation of cytochrome b(5) by urea was also analyzed. The first-order rate constants of heme transfer between cytochrome b(5) and apomyoglobin at 20 +/- 0.2 degrees C were 0.25 +/- 0.01 (wild type), 0.42 +/- 0.02 (Val61Tyr), 0.93 +/- 0.04 (Val61Glu), 2.88 +/- 0.01 (Val61His), and 3.88 +/- 0.02 h(-)(1) (Val61Lys). The crystal structure of Val61His was determined using the molecular replacement method and refined at 2.1 A resolution, showing that the imidazole side chain of His61 points away from the heme-binding pocket and extends into the solvent, the coordination distances from Fe to NE2 atoms of two axial ligands are approximately 0.6 A longer than the reported value, and the hydrogen bond network involving Val61, the heme propionates, and three water molecules no longer exists. We conclude that the conserved residue Val61 is located at one of the key positions, the "electrostatic potential" around the heme-exposed area and the hydrophobicity of the heme pocket are determinant factors modulating the redox potential of cytochrome b(5), and the hydrogen bond network around the exposed heme edge is also an important factor affecting the heme stability. Study holds ProTherm entries: 6554, 6555, 6556, 6557, 6558, 6560, 6561, 6562, 6563, 14323, 14324, 14325, 14326 Extra Details: electronic spectra; protein structur; first-order rate constant;,heme-binding pocket; electrostatic potential; hydrophobicity

Submission Details

ID: vbnLMZb5

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:32 p.m.

Version: 1

Publication Details
Xue LL;Wang YH;Xie Y;Yao P;Wang WH;Qian W;Huang ZX;Wu J;Xia ZX,Biochemistry (1999) Effect of mutation at valine 61 on the three-dimensional structure, stability, and redox potential of cytochrome b5. PMID:10508399
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 b5 P00171 CYB5_BOVIN
96.7 Cytochrome b5 P00172 CYB5_PIG
91.3 Cytochrome b5 P00170 CYB5_HORSE
91.3 Cytochrome b5 P00169 CYB5_RABIT
90.2 Cytochrome b5 P00167 CYB5_HUMAN
91.3 Cytochrome b5 P00173 CYB5_RAT
90.2 Cytochrome b5 P56395 CYB5_MOUSE
92.9 Cytochrome b5 P00168 CYB5_ALOSE