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

Substrate Binding Kinetics and its Role in the Cytochrome P450 Hydroxylation Sequence

The kinetics of the binding of type I and type II substrates to cytochrome P450LM has been investigated. Type I substrates are preferentially bound compared to type II compounds. The rate constants range between 103 and 105 [M-1 s-1], indicating possible interference with rate limiting steps. Substrates bind to the reduced cytochrome with considerably lower rate constants. The results are in favour of the sequential reaction mechanism. The solubilized enzyme preparation shows properties similar to the microsomal enzyme system

The Mode of Binding of Carbon Monoxide to Iron in Cytochrome P450 and Cytochrome P420. An Infrared Study

The infrared stretching vibrations (vc0 ) of the CO-complexes of cytochrome P450 and cytochrome P420 have been determined from the infrared difference spectra. The CO-complexes exhibit ir-bands at 1949 cm-1 and 1966 cm-1 with half widths of -17 cm·1 and - 20 cm·1 respectively. These results are con;ipared with the CO-stretching frequencies of other hemoproteins and discussed with respect to specific interactions of the CO-ligand with the protein moiety and to the ligand trans to CO in the cytochrome

Circular Dichroism of Partially Purified Cytochrome P450 from Rabbit Liver Microsomes

The heme-related circular dichroic bands of solubilized cytochrome P450 from rabbit liver microsomes and some of its liganded derivatives were measured in the Soret region. All P450 derivatives exhibit negative circular dichroic bands in the region of the Soret absorption. The wavelengths of the dichroic bands and their ellipticities vary with ligand substitution and the oxidation state of the iron. The results are compared with CD-data from other hemoproteins and discussed with respect to stereochemical conclusions concerning the geometry and the physicochemical character of the vicinity of the heme group with regard to results obtained from other studies

Differentiation Between Type I and Type II Substrate Binding to Cytochrome P450 by Temperature Studies

The binding reactions of type J and type II substances to cytochrome P450 solubilized from phenobarbital induced rat liver microsomes show different dependence on temperature. With increasing temperature the type II binding is lowered whereas the stability of type I substrate complexes increases. The binding enthalpies were calculated from the van\u27t Hoff plots and discussed in connection with the entropy and Gibbs energy of substrate binding to cytochrome P450. Our data on temperature dependence provide further evidence pointing to a difference between the binding sites of the two classes of substrates, and support the view that the type I binding site is located in a hydrophobic part of the cytochrome P450 molecule

Evidence of the Existence of a High Spin Low Spin Equilibrium in Liver Microsomal Cytochrome P450, and its Role in the Enzymatic Mechanism

In rabbit liver microsomal cytochrome P450 a high spin (S = = 5/2) low spin (S = 1/2) equilibrium has been proved to exist by recording temperature difference spectra in the Soret and in the visible region of the absorption spectrum of solubilized cytochrome P450. In the presence of type II substrates the predominantly low spin state of cytochrome P450 is maintained, only a very small shift to lower spin is observed. Ligands of the heme iron, such as cyanide and imidazole, pr9duce a pure low spin state and therefore in the presence of these ligands no temperature difference spectra can be obtained. In the presence of type I substrate, however, the spin equilibrium is shifted to the high spin state. The extent of this shift (1) depends on specific properties of the substrate and (2) it is generally relatively small, up to about 80/o in the case of substrates investigated so far