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

Cytochrome b5 (cyt b5) 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 alpha-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) 2B4 in a purified reconstituted system. Alanine was inserted at six positions in the membrane anchor of cyt b5. Insertion of alanine into an alpha-helix causes all amino acids at its carboxyl terminus to be rotated by 100 degrees . The ability of the alanine insertion mutants of cyt b5 to bind to cyt P450 2B4 was similar to that of the wild-type protein as was the ability of the mutant cyts b5 to stimulate the metabolism of the anesthetic, methoxyflurane. These results demonstrate that the C-terminal hydrophobic alpha-helix of cyt b5 does not interact with cyt P450 2B4 through a specific stereochemical fit of amino acid side chains, but rather through nonspecific interactions.