The Regulation of Surface Charged Residues on the Properties of Cytochrome B5

Overview

Journal J Protein Chem

Specialties Biochemistry
Chemistry

Date 2002 Jan 5

PMID 11760123

Authors

Y H Wang

Y Ren

W H Wang

Y Xie

Z X Huang

Affiliations

Soon will be listed here.

Abstract

To understand the roles of negatively surface charged residues, the cytochrome b5 (Cyt b5) E48A/D60A mutant was constructed. UV-visible and CD spectra confirmed that the mutation did not cause overall structural changes of the protein. The mutant presents an unexpected high stability toward the thermal and denaturant compared with the wild type Cyt b5, which shows that these surface charged residues can influence the interactions between the heme b group and the polypeptide chain. Functional properties were clarified through the electron transfer reactions between Cyt b5 and Cyt c. The driving force of the electron transfer reactions is conservative. Although the association constant of Cyt b5 E48A/D60A with Cyt c is much lower than that of the wild type Cyt b5, their electron transfer rate constants do not differ significantly. The results show that these surface charged residues play important roles in regulating both the stability and functional properties of Cyt b5.

References

Qian W, Sun Y, Wang Y, Zhuang J, Xie Y, Huang Z . The influence of mutation at Glu44 and Glu56 of cytochrome b5 on the protein's stabilization and interaction between cytochrome c and cytochrome b5. Biochemistry. 1998; 37(40):14137-50. DOI: 10.1021/bi9805036. View

Poulos T . Heme enzyme crystal structures. Adv Inorg Biochem. 1988; 7:1-36. View

Manyusa S, Whitford D . Defining folding and unfolding reactions of apocytochrome b5 using equilibrium and kinetic fluorescence measurements. Biochemistry. 1999; 38(29):9533-40. DOI: 10.1021/bi990550d. View

Hewson R, Newbold R, Whitford D . The expression of bovine microsomal cytochrome b5 in Escherichia coli and a study of the solution structure and stability of variant proteins. Protein Eng. 1993; 6(8):953-64. DOI: 10.1093/protein/6.8.953. View

Yao P, Xie Y, Wang Y, Sun Y, Huang Z, Xiao G . Importance of a conserved phenylalanine-35 of cytochrome b5 to the protein's stability and redox potential. Protein Eng. 1997; 10(5):575-81. DOI: 10.1093/protein/10.5.575. View

Salemme F . An hypothetical structure for an intermolecular electron transfer complex of cytochromes c and b5. J Mol Biol. 1976; 102(3):563-8. DOI: 10.1016/0022-2836(76)90334-x. View

Funk W, Lo T, Mauk M, Brayer G, MacGillivray R, Mauk A . Mutagenic, electrochemical, and crystallographic investigation of the cytochrome b5 oxidation-reduction equilibrium: involvement of asparagine-57, serine-64, and heme propionate-7. Biochemistry. 1990; 29(23):5500-8. DOI: 10.1021/bi00475a013. View

Sun Y, Wang Y, Yan M, Sun B, Xie Y, Huang Z . Structure, interaction and electron transfer between cytochrome b5, its E44A and/or E56A mutants and cytochrome c. J Mol Biol. 1999; 285(1):347-59. DOI: 10.1006/jmbi.1998.2295. View

Xue L, Wang Y, Xie Y, Yao P, Wang W, Qian W . Effect of mutation at valine 61 on the three-dimensional structure, stability, and redox potential of cytochrome b5. Biochemistry. 1999; 38(37):11961-72. DOI: 10.1021/bi990893b. View

10.

Pfeil W . Thermodynamics of apocytochrome b5 unfolding. Protein Sci. 1993; 2(9):1497-501. PMC: 2142466. DOI: 10.1002/pro.5560020914. View

11.

Caffrey M . Strategies for the study of cytochrome c structure and function by site-directed mutagenesis. Biochimie. 1994; 76(7):622-30. DOI: 10.1016/0300-9084(94)90139-2. View

12.

Mauk M, Reid L, Mauk A . Spectrophotometric analysis of the interaction between cytochrome b5 and cytochrome c. Biochemistry. 1982; 21(8):1843-6. DOI: 10.1021/bi00537a021. View

13.

Mauk A, Mauk M, MOORE G, Northrup S . Experimental and theoretical analysis of the interaction between cytochrome c and cytochrome b5. J Bioenerg Biomembr. 1995; 27(3):311-30. DOI: 10.1007/BF02110101. View

14.

Northrup S, Thomasson K, Miller C, Barker P, Eltis L, Guillemette J . Effects of charged amino acid mutations on the bimolecular kinetics of reduction of yeast iso-1-ferricytochrome c by bovine ferrocytochrome b5. Biochemistry. 1993; 32(26):6613-23. DOI: 10.1021/bi00077a014. View

15.

Wang Z, Wang Y, Wang W, Xue L, Wu X, Xie Y . The effect of mutation at valine-45 on the stability and redox potentials of trypsin-cleaved cytochrome b5. Biophys Chem. 2000; 83(1):3-17. DOI: 10.1016/s0301-4622(99)00119-2. View

16.

Estabrook R, Hildebrandt A, Baron J, NETTER K, Leibman K . A new spectral intermediate associated with cytochrome P-450 function in liver microsomes. Biochem Biophys Res Commun. 1971; 42(1):132-9. DOI: 10.1016/0006-291x(71)90372-x. View

17.

Bernardi P, Azzone G . Cytochrome c as an electron shuttle between the outer and inner mitochondrial membranes. J Biol Chem. 1981; 256(14):7187-92. View

18.

Caffrey M, Cusanovich M . Site-specific mutagenesis studies of cytochromes c. Biochim Biophys Acta. 1994; 1187(3):277-88. DOI: 10.1016/0005-2728(94)90001-9. View

19.

Davidson V . What controls the rates of interprotein electron-transfer reactions. Acc Chem Res. 2000; 33(2):87-93. DOI: 10.1021/ar9900616. View

20.

Sannes L, HULTQUIST D . Effects of hemolysate concentration, ionic strength and cytochrome b5 concentration on the rate of methemoglobin reduction in hemolysates of human erythrocytes. Biochim Biophys Acta. 1978; 544(3):547-54. DOI: 10.1016/0304-4165(78)90329-x. View