INTERACTIONS OF CYTOCHROME-P450 2B4 WITH NADPH-CYTOCHROME P450 REDUCTASE STUDIED BY FLUORESCENT-PROBE

A new method for monitoring the formation of the cytochrome P450 compl exes with NADPH-cytochrome P450 reductase (NCPR) is introduced. The me thod is based on the quenching of fluorescence of NCPR labelled with t hylamino-3-(4'-maleimidilphenyl)-4-methylcoumarin maleimide (CPM). In a monomerized soluble reconstituted system in the absence of phospholi pid, cytochrome P450 2B4 and NCPR(cpm) were shown to form 1:1 complexe s with a K-d of 0.038 mu M. Formation of the complex follows the kinet ics of reversible second order transition with k(on) = 6.5 . 10(5) M(- 1)s(-1). Application of high hydrostatic pressure induces dissociation of the complex (Delta V degrees = -65 mL/mol). Succinylation of the h emoprotein increases the value of K-d to 0.5 mu M primarily by decreas ing k(on). In contrast to what was shown for intact 2B4, rising pressu re does not take apart succinylated hemoprotein and NCPR(cpm) molecule s, but causes some internal transition in their complex that diminishe s the quenching. This transition is characterised by a very large volu me change (Delta V degrees = -155 mL/mol). The following conclusions w ere drawn: 1) a molecule of 2B4 contains two distinct contact regions involved in the interactions with NCPR. Only one of these regions is p olar and highly hydrated in unbound hemoprotein; 2) interactions of th e polar regions of 2B4 and NCPR are necessary to bring CPM-labelled cy steine of NCPR in short distance of the heme of 2B4; and 3) some of th e lysine residues located in the proximity of the polar binding region are apparently involved in the formation of the internal salt bridges in the molecule of 2B4.