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

Abstract

Cytochrome P450 2B4 lacking amino acids 2-27, CYP2B4 (delta2-27), was mutated at position 250 and expressed in E. coli fused to glutathione S-transferase. Expression of the E250S variant (holo- plus apoenzyme) proceeded to an extent comparable with that of CYP2B4 (delta2-27), 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 (delta2-27) 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 (delta2-27); introduction of an alpha-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.