Heme maintains catalytically active structure of cytochrome P‐450

Abstract

Treatment of purified cytochrome P-450 LM2 and its liposome-bound form with hydrogen peroxide led to complete destruction of the P-450 heme. The apoenzyme thus produced could be reconstituted to catalytically active cytochrome P-450 by incubation with hemin, the reconstitution efficiency being 50% for the soluble enzyme and 80% for the liposome-bound enzyme. The removal of heme from the soluble hemoprotein resulted in a 3-fold decrease in the efficiency of its incorporation into sonicated liposomes. The contents of 5 secondary structure forms in the native, apoand reconstituted holoenzymes were estimated from their circular dichroism spectra. It was thus found that the helix content increased from 34% to 60% upon removal of the heme from the native enzyme. We suggest that the increase in the helix content leads to a reduction of the incorporation efficiency into liposomal membranes.

Determination of membrane‐bound fragments of cytochrome P‐450 2B4

“…Thus, our data make it evident that only the N-terminal sequence 1-20 of the cytochrome P-450 2B4 molecule is deeply embedded in the membrane or even penetrates the membrane. This conclusion is in agreement with results of the comprehensive proteolysis proteoliposomes containing cytochrome P-450 2B4 [33]. We found that only the N-terminal 21 -amino-acid fragment remained bound to the liposomes.…”

Electron transfer in the complex of membrane-bound human cytochrome P450 3A4 with the flavin domain of P450BM-3: The effect of oligomerization of the heme protein and intermittent modulation of the spin equilibrium

“…Destruction of the porphyrin ring by H 2 O 2 is known as an efficient and gentle approach to deplete cytochromes P450 completely of the heme group [38, 53, 54]. Therefore, in order to estimate the efficiency of FRET from BODIPY to the heme chromophore we employed the heme depletion (bleaching) of CYP3A4(C468)-BODIPY in the presence of H 2 O 2 .…”

ROLE OF CYTOCHROMEB5IN MODULATING PEROXIDE-SUPPORTED CYP3A4 ACTIVITY: EVIDENCE FOR A CONFORMATIONAL TRANSITION AND CYTOCHROME P450 HETEROGENEITY

“…Removal of the heme from CYP3A4 and preparation of the apo-protein was performed by treatment with H 2 O 2 (Uvarov et al, 1990;Pikuleva et al, 1992). We implemented very mild conditions of treatment to prevent any peroxidative damage of the protein.…”

“…Determination of the kinetics of CYP3A4 heme depletion in the presence of H 2 O 2 was done using conditions similar to those previously described (Uvarov et al, 1990;Pikuleva et al, 1992). The reaction was carried out at 25°C in 100 mM Hepes buffer, pH 7.4, in a 1-ml semi-microspectrophotometric cell with constant stirring.…”