4L77

P450cin Active Site Water: Implications for Substrate Binding and Solvent Accessibility

Summary for 4L77

• Entry DOI: 10.2210/pdb4l77/pdb
• Related: 4L6G 4LHT
• Descriptor: P450cin, PROTOPORPHYRIN IX CONTAINING FE, 1,3,3-TRIMETHYL-2-OXABICYCLO[2.2.2]OCTANE, ... (7 entities in total)
• Functional Keywords: p450, heme, monooxygenase, cindoxin, oxidoreductase
• Biological source: Citrobacter braakii
• Total number of polymer chains: 2
• Total formula weight: 91727.94

Authors

Madrona, Y.,Poulos, T.L. (deposition date: 2013-06-13, release date: 2013-07-24, Last modification date: 2024-02-28)

Primary citation

Madrona, Y.,Hollingsworth, S.A.,Khan, B.,Poulos, T.L.
P450cin active site water: implications for substrate binding and solvent accessibility.
Biochemistry, 52:5039-5050, 2013

PubMed 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.

PubMed: 23829586
DOI: 10.1021/bi4006946

Experimental method

X-RAY DIFFRACTION (1.379 Å)