Amino acid residue 250 has a functional role in the assembly of rabbit liver microsomal cytochrome P450 2B4

• Published in: Biochemistry and molecular biology international 1998-05, Vol.44 (6), p.1147-1155
• Main Authors: Schulze, Johannes, Lehnerer, Michael, Lewis, David F. V., Hlavica, Peter
• Format: Article
• Language: English
• Subjects: Animals; Aryl Hydrocarbon Hydroxylases; CYP2B4 (Δ2‐27); Cytochrome P-450 Enzyme System - biosynthesis; Cytochrome P-450 Enzyme System - genetics; DNA, Complementary - genetics; glutamate‐250; Glutamic Acid - metabolism; Glutathione Transferase - genetics; heme incorporation; Microsomes, Liver - enzymology; Mutagenesis, Site-Directed; Protein Structure, Secondary; Rabbits; salt bridge; Steroid Hydroxylases - biosynthesis; Steroid Hydroxylases - genetics
• ISSN: 1521-6543; 1039-9712; 1521-6551
• DOI: 10.1080/15216549800202232

Cytochrome P450 2B4 lacking amino acids 2‐27, CYP2B4 (Δ2‐27), was mutated at position 250 and expressed in E.coli fused toglutathione S‐transferase. Expression of the E250S variant (holo‐plus apoenzyme) proceeded to an extent comparable with that of CYP2B4 (Δ2‐27), while the protein level of the E250P mutant averaged 42% that of the control pigment. Comparison of these data with the corresponding reduced CO difference spectra of the various CYP2B4 (Δ2‐27) forms revealed that, in the control and E250S preparations, about 90% and 44%, respectively, of the total amount of hemoprotein present existed in the form of holoenzyme, whereas the E250P derivative failed to produce a reduced carbonyl complex. Thus, replacement of the negatively charged E250 with an uncharged, polar serine residue substantially hampered assembly of CYP2B4 (Δ2‐27); introduction of an α‐helix‐disrupting proline completely blocked the formation of holoenzyme. These phenomena suggested that the negative charge of E250, residing in the putative G helix, underwent pairing with some positively charged group, possibly H285 located in the I helix. Deletion of the negative charge obviously perturbed the active‐site geometry such as to affect both the incorporation and/or retention of the heme ligand and the spectral binding of substrates such as hexobarbital.