Role of the interdomain linker probed by kinetics of CO ligation to an endothelial nitric oxide synthase mutant lacking the calmodulin binding peptide (residues 503-517 in bovine).
Oxygenase and reductase domains in nitric oxide synthase are linked by a peptide region that binds calmodulin. Here we study the effects of modifying the length of the interdomain linker in a deletion mutant lacking 15 amino acids (residues 503-517) in bovine eNOS. The kinetics of CO ligation with the mutant were determined in the presence and absence of tetrahydrobiopterin and arginine and compared with the CO binding kinetics of wild-type eNOS and the eNOS oxygenase domain. In the mutant, electron flow is interrupted. The association kinetics of CO with both mutant and wild-type eNOS can be approximated with two kinetic phases, but the relative proportions change in the mutant. Both the abrogation of electron flow in the mutant and the differences in CO binding may be explained by an alteration in the docking of the FMN domain to the heme domain. We propose that the calmodulin binding residues form a helix that is critical for the proper alignment of the adjacent reductase and oxygenase domains within the active eNOS dimer in achieving proper electron transfer between them.
Zemojtel
Tomasz
T
Departments of Pharmacology I and Medicine I, University of Freiburg, Germany.
Scheele
Jurgen S
JS
Martásek
Pavel
P
Masters
Bettie Sue Siler
BS
Sharma
Vijay S
VS
Magde
Douglas
D
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000737:chemistry*; D002248:Carbon Monoxide / Q000737:chemistry*; D002417:Cattle; D019281:Dimerization; D004583:Electrons; D017353:Gene Deletion; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D052250:Nitric Oxide Synthase Type III; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014466:Ultraviolet Rays
D002147:Calmodulin; D010455:Peptides; D006418:Heme; D002248:Carbon Monoxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III