The α-helical membrane spanning domain of cytochrome b 5 interacts with cytochrome P450 via nonspecific interactions

• Published in: Biochimica et biophysica acta. General subjects Vol. 1674; no. 3; pp. 319 - 326
• Main Authors: Mulrooney, Scott B., Meinhardt, David R., Waskell, Lucy
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.11.2004
• Subjects: Cytochrome b5; Cytochrome P450 2B4; Membrane protein; Mutagenesis; Protein–protein interaction; Membrane protein; Mutagenesis; Cytochrome b 5; Cytochrome P450 2B4; Protein–protein interaction
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2004.08.001

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 α-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 b 5 and cytochrome P450 (cyt P450) 2B4 in a purified reconstituted system. Alanine was inserted at six positions in the membrane anchor of cyt b 5. Insertion of alanine into an α-helix causes all amino acids at its carboxyl terminus to be rotated by 100°. The ability of the alanine insertion mutants of cyt b 5 to bind to cyt P450 2B4 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 α-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.