Pseudomonas putida cytochrome P-450. The effect of complexes of the ferric hemeprotein on the relaxation of solvent water protons.
With pulsed nuclear magnetic resonance techniques, the effects of various complexes of ferric cytochrome P-450 on the relaxation rate of bulk solution water protons have been determined. For the camphor, metyrapone, and 4-phenylimidazole complexes, the experimental results are consistent with outer sphere relaxation effects. However, for the substrate-free enzyme, the magnitude and temperature dependence of the paramagnetic relaxation effects indicate the presence of exchangeable protons in the coordination sphere of the heme iron atom. The exchange rate (9.3 x 10(4) S-1 at 25 degrees) and the thermodynamic activation parameters for the exchange process are very similar to those of acid metmyoglobin and acid methemoglobin, suggesting that a water molecule, and not an amino acid residue of the protein, coordinates to the ferric cation of the enzyme in the absence of added substrate or ligands. From the equations appropriate for coordination sphere protons, the distance between these protons and the ferric heme cation was evaluated as 2.1 A, which further supports the interpretation. These experimental results demonstrate that the solvent accessibility of the ferric cation of substrate-free cytochrome P-450 is significantly reduced by the binding of substrate or nitrogenous ligands to the hemeprotein.
D001665:Binding Sites; D002164:Camphor; D003577:Cytochrome P-450 Enzyme System; D007501:Iron / Q000032:analysis; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology*; D014867:Water
D014867:Water; D002164:Camphor; D003577:Cytochrome P-450 Enzyme System; D007501:Iron