The α-helical membrane spanning domain of cytochrome b5 interacts with cytochrome P450 via nonspecific interactions

Cytochrome b(5) (cyt b(5)) is an amphipathic membrane-bound heme protein found in the endoplasmic reticulum of eukaryotes. It consists of three domains, an N-terminal cytosolic, hydrophilic domain containing the heme, a short flexible linker and an a-helical membrane-spanning domain. This study investigated whether there are specific side chain helix-helix packing interactions between the COOH-terminal membrane anchor of cyt b5 and cytochrome P450 (cyt P450) 2134 in a purified reconstituted system. Alanine was inserted at six positions in the membrane anchor of cyt b(5). Insertion of alanine into an a-helix causes all amino acids at its carboxyl terminus to be rotated by 100degrees. The ability of the alanine insertion mutants of cyt b(5) to bind to cyt P450 2134 was similar to that of the wild-type protein as was the ability of the mutant cyts b(5) to stimulate the metabolism of the anesthetic, methoxyflurane. These results demonstrate that the C-terminal hydrophobic alpha-helix of cyt b(5) does not interact with cyt P450 2B4 through a specific stereochemical fit of amino acid side chains, but rather through nonspecific interactions. (C) 2004 Elsevier B.V All rights reserved.