The comparative study on the solution structures of the oxidized bovine microsomal cytochrome b5 and mutant V45H.
A comparative study on the solution structures of bovine microsomal cytochrome b5 (Tb5) and the mutant V45H has been achieved by 1D and 2D 1H-NMR spectroscopy to clarify the differences in the solution conformations between these two proteins. The results reveal that the global folding of the V45H mutant in solution is unchanged, but the subtle changes exist in the orientation of the axial ligand His39, and heme vinyl groups. The side chain of His45 in V45H mutant extends to the outer edge of the heme pocket leaving a cavity at the site originally occupied by the inner methyl group of Val45 residue. In addition, the imidazole ring of axial ligand His39 rotates counterclockwise by approximately 3 degrees around the His-Fe-His axis, and the 4-heme vinyl group turns to the space vacated by the removed side chain due to the mutation. Furthermore, the helix III of the heme pocket undergoes outward displacement, while the linkage between helix II and III is shifted leftward. These observations are not only consistent with the pattern of the pseudocontact shifts of the heme protons, but also well account for the lower stability of V45H mutant against heat and urea.
State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, 200032 PR China.
Qi
Q
Zhang
Chunyang
C
Cao
Zhi-Qiang
ZQ
Wang
Yun-Hua
YH
Wang
Houming
H
Wu
Zhong-Xian
ZX
Huang
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D002417:Cattle; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics; D006418:Heme / Q000737:chemistry*; D008861:Microsomes / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D017354:Point Mutation / Q000235:genetics*; D011489:Protein Denaturation / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics
D006418:Heme; D015786:Cytochromes b5