title: P450cin active site water: implications for substrate binding and solvent accessibility.
abstract: In P450cin, Tyr81, Asp241, Asn242, two water molecules, and the substrate participate in a complex H-bonded network. The role of this H-bonded network in substrate binding and catalysis has been probed by crystallography, spectroscopy, kinetics, isothermal titration calorimetry (ITC), and molecular dynamics. For the Y81F mutant, the substrate binds about 20-fold more weakly and Vmax decreases by about 30% in comparison to WT. The enhanced susceptibility of the heme to H₂O₂-mediated destruction in Y81F suggests that this mutant favors the open, low-spin conformational state. Asn242 H-bonds directly with the substrate, and replacing this residue with Ala results in water taking the place of the missing Asn side chain. This mutant exhibits a 70% decrease in activity. Crystal structures and molecular dynamics simulations of substrate-bound complexes show that the solvent has more ready access to the active site, especially for the N242A mutant. This accounts for about a 64% uncoupling of electron transfer from substrate hydroxylation. These data indicate the importance of the interconnected water network on substrate binding and on the open/closed conformational equilibrium, which are both critically important for maintaining high-coupling efficiency.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900, United States.
authors.forename: Yarrow
authors.initials: Y
authors.lastname: Madrona
authors.forename: Scott A
authors.initials: SA
authors.lastname: Hollingsworth
authors.forename: Bushra
authors.initials: B
authors.lastname: Khan
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Poulos
mesh_terms: D019943:Amino Acid Substitution; D001216:Asparagine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002954:Citrobacter / Q000201:enzymology*; D003511:Cyclohexanols / Q000737:chemistry / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport; D000077591:Eucalyptol; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D006900:Hydroxylation; D007700:Kinetics; D008958:Models, Molecular; D039821:Monoterpenes / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013499:Surface Properties; D014867:Water / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003511:Cyclohexanols; D039821:Monoterpenes; D050505:Mutant Proteins; D011994:Recombinant Proteins; D014867:Water; D001216:Asparagine; D003577:Cytochrome P-450 Enzyme System; C465212:cytochrome P-450 176A; D000077591:Eucalyptol