Methanol-Induced Unfolding and Refolding of Cytochrome b5 and Its P40V Mutant Monitored by UV-Visible, CD, and Fluorescence Spectra

  title={Methanol-Induced Unfolding and Refolding of Cytochrome b5 and Its P40V Mutant Monitored by UV-Visible, CD, and Fluorescence Spectra},
  author={Z. Q. Wang and Y. H. Wang and Wen Qian and H. H. Wang and Lijuan Chunyu and Y. Xie and Z. X. Huang},
  journal={Journal of Protein Chemistry},
In order to illustrate the structural importance of proline-40 of cytochrome b5 (Cyt b5), the P40V mutant gene was constructed. Unfolding and refolding of Cyt b5 induced by methanol was investigated by means of the UV-visible spectrum, circular dichroism, and the fluorescence spectrum. Methanol denaturation of Cyt b5 is a cooperative process, that is, the heme group dissociates from the heme pocket accompanied by unfolding of the polypeptide chain both in the secondary and tertiary structures… 
Proline-40 is essential to maintaining cytochrome b^5's stability and its electron transfer with cytochrome c
These studies provided a good example that property and functional changes of a protein do not necessarily require large overall structural alterations; in most cases, only perturbations on the local conformations are sufficient to induce significant changes in protein′s properties and functions.
How membrane surface affects protein structure
It is concluded that the negatively charged membrane surface can serve as a moderately denaturing agent in the cell and the effect of the membrane field on the protein structure must be taken into account.
Mechanistic basis of electron transfer to cytochromes p450 by natural redox partners and artificial donor constructs.
  • P. Hlavica
  • Biology, Chemistry
    Advances in experimental medicine and biology
  • 2015
Deeper insight into the mechanistic basis of the redox machinery will permit optimization of redox processes via directed evolution and DNA shuffling and help obviate the tedious reconstitution procedure and induces novel activities.


Conformational states in denaturants of cytochrome c and horseradish peroxidases examined by fluorescence and circular dichroism.
Steady-state fluorescence and circular dichroism (CD) were used to examine the unfolding in denaturants of recombinant cytochrome c peroxidase and horseradish peroxids in their ferric forms, indicating that HRP is kinetically much more stable than CCP(MI.
The methanol-induced globular and expanded denatured states of cytochrome c: a study by CD fluorescence, NMR and small-angle X-ray scattering.
Methanol-induced conformational transitions of cytochrome c(cyt c) at acidic pH values were investigated with a combined use of far and near-UV CD, fluorescence, NMR spectroscopy and small-angle X-ray scattering showing that the I(M) state has no specific tertiary structure but has a secondary structural content and tryptophan environment similar to those in the native state.
Structure, interaction and electron transfer between cytochrome b5, its E44A and/or E56A mutants and cytochrome c.
These studies, by means of a series of techniques, provide conclusive results that the interaction between cytochrome b5 and cy tochrome c is electrostatically guided, and, more importantly, that both Glu44 and Glu56 participate in the electron transfer reaction.
The influence of mutation at Glu44 and Glu56 of cytochrome b5 on the protein's stabilization and interaction between cytochrome c and 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.
Mutagenic, electrochemical, and crystallographic investigation of the cytochrome b5 oxidation-reduction equilibrium: involvement of asparagine-57, serine-64, and heme propionate-7.
Spectroelectrochemical studies of a mutant in which Ser-64 has been changed to an alanyl residue demonstrate that this protein has a reduction potential that is 7 mV lower than that of the wild-type protein; moreover, conversion of the heme propionate groups to the corresponding methyl esters increases the potential by 67 mV.
The stability of holomyoglobin is determined by heme affinity.
Many previous studies of the denaturation of intact heme proteins need to be reevaluated in terms of heme affinity and measurements with apoproteins are required for unambiguous determinations of the stability of globin structures.
The function of tyrosine 74 of cytochrome b5.
The Tyr74Lys mutant exhibits a normal redox potential and spectroscopic properties identical to those of the wild type protein, and Heme transfer experiments demonstrate that heme spontaneously dissociates 6 times faster from the mutant than the wildtype protein.