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 complexes with NADPH-cytochrome P450 reductase (NCPR) is introduced. The method is based on the quenching of fluorescence of NCPR labelled with 7-ethylamino-3-(4'-maleimidilphenyl)-4-methylcoumarin maleimide (CPM). In a monomerized soluble reconstituted system in the absence of phospholipid, cytochrome P450 2B4 and NCPRcpm were shown to form 1:1 complexes with a Kd of 0.038 microM. Formation of the complex follows the kinetics 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 hemoprotein increases the value of Kd to 0.5 microM primarily by decreasing k(on). In contrast to what was shown for intact 2B4, rising pressure does not take apart succinylated hemoprotein and NCPRcpm molecules, but causes some internal transition in their complex that diminishes the quenching. This transition is characterised by a very large volume change (delta V degrees = -155 mL/mol). The following conclusions were drawn: 1) a molecule of 2B4 contains two distinct contact regions involved in the interactions with NCPR. Only one of these regions is polar and highly hydrated in unbound hemoprotein; 2) interactions of the polar regions of 2B4 and NCPR are necessary to bring CPM-labelled cysteine of NCPR in short distance of the heme of 2B4; and 3) some of the lysine residues located in the proximity of the polar binding regions are apparently involved in the formation of the internal salt bridges in the molecule of 2B4.
Davydov
D R
DR
Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow, Russia.
Knyushko
T V
TV
Kanaeva
I P
IP
Koen
Y M
YM
Samenkova
N F
NF
Archakov
A I
AI
Hui Bon Hoa
G
G
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D003374:Coumarins / Q000737:chemistry*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D011817:Rabbits; D013250:Steroid Hydroxylases / Q000378:metabolism*
D003374:Coumarins; D005456:Fluorescent Dyes; C030860:N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C050356:steroid 15-alpha-hydroxylase; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase