The influence of mutation at Glu44 and Glu56 of cytochrome b5 on the protein's stabilization and interaction between cytochrome c and cytochrome b5.
To characterize the roles played by Glu44 and Glu56 of cytochrome b5 in the formation of the electrostatic complex between cytochrome c and cytochrome b5, the Glu44, Glu56, or both sites were changed to alanine by site-directed mutagenesis. The influence of these two residues on the protein stability was probed by investigating the kinetic behaviors of protein denaturation in urea or upon heating and the heme-transfer reactions between apo-myoglobin and the variants of cytochrome b5. It has been found that when the Glu44 and/or Glu56 are mutated to alanine, the protein stability increases slightly due to the fact that the hydrophilic residue is changed to a hydrophobic one, resulting in the two pairs of helices surrounding the heme taking a more compact conformation. The difference in voltammetric behavior of cytochrome c, cytochrome b5, and its three mutants, Cyt b5 E44A, E56A, and E44/56A, alone and in 1:1 protein complexes demonstrates that both Glu44 and Glu56 of cytochrome b5 take part in the electrostatic interaction with cytochrome c. The entropy changes, DeltaS degreesrc and enthalpy changes, DeltaH degrees, derived from the temperature dependence of the formal reduction potentials of each protein in different protein systems suggest that, because of the mutual interaction with cytochrome c, cytochrome b5 mutants, especially the E44A-containing mutants, in the protein complexes suffer greater conformational changes upon reduction than that of the wild type. The variation of these thermodynamic parameters indicates that the strength of mutual interactions between cytochrome c and cytochrome b5 or its mutants has the following order: Cyt c/Cyt b5 > Cyt c/Cyt b5 E56A > Cyt c/Cyt b5 E44A > Cyt c/Cyt b5 E44/56A.
Department of Chemistry, Fudan University, Shanghai, China.
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W
Qian
Y L
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Sun
Y H
YH
Wang
J H
JH
Zhuang
Y
Y
Xie
Z X
ZX
Huang
D000818:Animals; D001059:Apoproteins / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D015786:Cytochromes b5 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004563:Electrochemistry; D004735:Energy Transfer / Q000235:genetics; D019277:Entropy; D004795:Enzyme Stability / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics*; D006418:Heme / Q000378:metabolism; D006736:Horses; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000378:metabolism; D010084:Oxidation-Reduction; D012996:Solutions; D013696:Temperature; D014508:Urea / Q000378:metabolism
D001059:Apoproteins; D003574:Cytochrome c Group; D009211:Myoglobin; D012996:Solutions; C010857:apomyoglobin; D018698:Glutamic Acid; D006418:Heme; D014508:Urea; D015786:Cytochromes b5