The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.
Cytochrome b(5) (cyt b(5)) is a 15-kDa amphipathic protein with a cytosolic amino-terminal catalytic heme domain, which is anchored to the microsomal membrane by a hydrophobic transmembrane alpha-helix at its carboxyl terminus. These two domains are connected by an approximately 15-amino acid linker domain, Ser(90)-Asp(104), which has been modified by site-directed mutagenesis to investigate whether the length or sequence of the linker influences the ability of cyt b(5) to bind ferric cytochrome P450 2B4 and donate an electron to oxyferrous (cyt P450 2B4), thereby stimulating catalysis. Because shortening the linker by 8 or more amino acids markedly inhibited the ability of cyt b(5) to bind cyt P450 2B4 and stimulate catalysis by this isozyme, it is postulated 7 amino acids are sufficient to allow a productive interaction. All mutant cyts b(5) except the protein lacking the entire 15-amino acid linker inserted normally into the microsomal membrane. Alternatively, lengthening the linker by 16 amino acids, reversing the sequence of the amino acids in the linker, and mutating conserved linker residues did not significantly alter the ability of cyt b(5) to interact with cyt P450 2B4. A model for the membrane-bound cyt b(5)-cyt P450 complex is presented.
Clarke
Thomas A
TA
Centre of Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
Im
Sang-Choul
SC
Bidwai
Anil
A
Waskell
Lucy
L
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry*; D001224:Aspartic Acid / Q000737:chemistry; D002384:Catalysis; D002417:Cattle; D018360:Crystallography, X-Ray; D000072467:Cytochrome P450 Family 2; D015786:Cytochromes b5 / Q000737:chemistry*; D018076:DNA, Complementary / Q000378:metabolism; D019992:Databases as Topic; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D006418:Heme / Q000737:chemistry; D006801:Humans; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D015233:Models, Statistical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013053:Spectrophotometry
D018076:DNA, Complementary; D020033:Protein Isoforms; D001224:Aspartic Acid; D006418:Heme; D012694:Serine; D015786:Cytochromes b5; D007501:Iron; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit)