Crystal structure of recombinant trypsin-solubilized fragment of cytochromeb5 and the structural comparison with Val61His mutant

Abstract

The crystal structure of the recombinant trypsin-solubilized fragment of the microsomal cytochrome b(5) from bovine liver has been determined at 1.9 A resolution and compared with the reported crystal structure of the lipase-solubilized fragment of the membrane protein cytochrome b(5). The two structures are similar to each other. However, some detailed structural differences are observed: the conformation of the segment Asn16-Ser20 is quite different, some helices around the heme and some segments between the helices are shifted slightly, the heme is rotated about the normal of the mean plane of heme, one of the propionates of the heme exhibits a different conformation. The average coordination distances between the iron and the two nitrogen atoms of the imidazole ligands are the same in the two structures. Most of the structural differences can be attributed to the different intermolecular interactions which result from the crystal packing. The wild-type protein structure is also compared with its Val61His mutant, showing that the heme binding and the main chain conformations are basically identical with each other except for the local area of the mutation site. However, when Val61 is mutated to histidine, the large side chain of His61 is forced to point away from the heme pocket toward the solvent region, disturbing the micro-environment of the heme pocket and influencing the stability and the redox potential of the protein.

X‐ray crystallography, CD and kinetic studies revealed the essence of the abnormal behaviors of the cytochrome b5 Phe35→Tyr mutant

“…Single crystals of the Phe35fiTyr mutant of trypsin-solubilized bovine liver microsomal cytochrome b 5 (Tb 5 ) were grown by the vapor diffusion method in hanging drops containing 10 mgAEmL )1 protein solution in 3.1-3.2 M phosphate buffer (pH 7.5) at 20°C. This is similar to the crystallizing condition used for wild-type Tb 5 [21] and lipase-solubilized bovine liver microsomal cytochrome b 5 (Lb 5 ) [22]. The typical size of the single crystals was 0.6 · 0.5 · 0.3 mm.…”

“…The typical size of the single crystals was ≈ 0.6 × 0.5 × 0.3 mm. Crystals of wild‐type T b 5 [21] and the T b 5 Val61→His mutant [23] are isomorphous belonging to the monoclinic space group C2 with the following unit cell parameters: a = 70.71 Å, b = 40.39 Å, c = 39.30 Å and β = 111.72°.…”

Desaturases fused to their electron donor

“…Despite the growing number of sequences of plant origin [115], a differentiation in microsomal and mitochondrial forms has not yet been made. They can be released from membranes by proteolysis, and the fragments from both, the microsomal [16,157] and the outer mitochondrial membrane [116], have been crystallized and subjected to X-ray analysis with resolutions between 1.5 and 2.7 Å. The hydrophilic domains of the microsomal and mitochondrial isoenzymes are roughly 100 amino acids long and share about 60% sequence identity.…”

Structures of V45E and V45Y mutants and structure comparison of a variety of cytochrome b5 mutants

“…The crystal structures of both the lipase-solubilized fragment and the recombinant trypsin-solubilized fragment of cytochrome b 5 were determined at high resolution (Durley & Mathews, 1996;Wu et al, 2000), which provide the detailed structural basis for studying the structure±function relationship. Recently, the technique of site-directed mutagenesis was used to investigate the speci®c role played by various key residues of cytochrome b 5 .…”