Hepatic Cytochrome P-450. A Proton Magnetic Relaxation Study of Microsomal, Solubilized and Partially Reconstituted Enzyme System

The longitudiJ:ial proton magnetic relaxation times, Ti, were measured from -5 to 40 °c for microsomal, solubilized and reconstituted cytochrome P-450 obtained from phenobarbital-induced rat livers. The paramagnetic contribution to the rates was derived by subtraction of the rates measured on dithionite-CO-reduced samples. The same values were obtained for microsomal P-450 on reduction with NADPH. PMR titratio.n by KCN yielded a dissociation constant of about 30 mM. This is three orders of magnitude larger than for metmyoglobin. It is concluded that the measured PMR rates are most likely due to the P-450 (and P-420) haem-iron while the 300/o non-haem iron found in both the microsomal and s olubilized P-450 is .ineffective for the PMR rates. These rates increase several times on isotopic dilution (D20 for H20) with the microsomes and diminish for the solubilized samples. Microsomes show 170/o residual, encaged, H20. Most of their paramagnetic PMR rate is due to the parama.gnetic iron located on the outside of microsomes. This is demonstrated by measurements with deuterated samples to which 190/o H20 had been added. Hence, the solubilized P-450 is homogeneous regarding PMR, but the microsomes are not