Abstract

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.

Highlights

  • The relaxation of proton spins in the coordination sphere of a paramagnetic ion in solution is described quantitatively by the Solomon-Bloembergen equations [27, 28] (Equations 1 and 2).In Equations 1 and 2, T, and T, are the longitudinal and transverse relaxation times, respectively; y, is the proton gyromagnetic ratio; /Y, the Bohr magneton, and S and g are the total spin and g value, respectively, of the paramagnetic ion.The distance between the ion and the proton is I^

  • For the complexes of ferric cytochrome P-450 studied, values of S were determined from EPR measurements at temperatures near that of liquid helium, where the substratefree form is predominantly low spin (S = l/2), while the camphor-bound form is predominantly high spin (S = 5/2)

  • EDTA solution) or a sample of cytochrome P-450 that was essentially 100% low spin; the low spin signal accounts for approximately 5% of the total cytochrome P-450 concentration of the sample

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Summary

Introduction

The relaxation of proton spins in the coordination sphere of a paramagnetic ion in solution is described quantitatively by the Solomon-Bloembergen equations [27, 28] (Equations 1 and 2).In Equations 1 and 2, T,, and T,, are the longitudinal and transverse relaxation times, respectively; y, is the proton gyromagnetic ratio; /Y, the Bohr magneton, and S and g are the total spin and g value, respectively, of the paramagnetic ion.The distance between the ion and the proton is I^. The relaxation of proton spins in the coordination sphere of a paramagnetic ion in solution is described quantitatively by the Solomon-Bloembergen equations [27, 28] (Equations 1 and 2). In Equations 1 and 2, T,, and T,, are the longitudinal and transverse relaxation times, respectively; y, is the proton gyromagnetic ratio; /Y, the Bohr magneton, and S and g are the total spin and g value, respectively, of the paramagnetic ion. The distance between the ion and the proton is I^. A is the hyperfine coupling constant between the two spins, and h is Planck’s constant divided by 2 H. The Larmor precession frequency of the proton is w,, while that of the paramagnetic ion is us. The correlation time for the dipolar interaction, T,, is given by and the correlation given by

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