JBC, Vol. 250, Issue 16, 6445-6451, Aug, 1975
Pseudomonas putida cytochrome P-450. The effect of complexes of the ferric hemeprotein on the relaxation of solvent water protons
B. W. Griffin and J. A. Peterson
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.
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