Electrostatic interactions between cytochrome P-450 LM2 and NADPH-cytochrome P-450 reductase.
At pH 8.2 and a 100-fold molar excess of the amino group specific label 2-methoxy-5-nitrotropone (MNT) over protein 2 mol MNT/mol P-450 LM2 were bound, which caused a 50% decrease in the overall activity due to a decreased electron transfer rate from reductase to the hemoprotein. However, different from FITC modification, which produces the same effects, the label is not selectively bound to the alpha-amino group and to lysine 384, but reacts with lysines in positions 49, 100, 139, 144, 251, 384 and 433. The decrease in the overall activity and reduction rate thereby correlates with a relative increase in the modification of lysines 139, 144, 251 and 384. Thus, besides lysine 384 the epsilon-amino groups of lysines 139, 144 and 251 are further candidates for participation in the interaction with reductase. This finding supports our model of charge-pair contacts between P-450 and reductase, where amino groups of P-450 LM2 form salt bridges to carboxylic groups of reductase. The decrease of reductase supported P-450 reduction velocity in microsomes at high salt concentration (I greater than 222 mM) indicates the dominant electrostatic character of P-450/reductase interaction. Based on these results and data from the literature a model of membrane topography of P-450 LM2 has been proposed. Extension of the charge pair interaction model to interaction mechanisms of other P-450 isoenzymes and forms with their respective electron donors is discussed.
Bernhardt
R
R
Central Institute of Molecular Biology, Academy of Sciences of the GDR, Berlin-Buch.
Kraft
R
R
Otto
A
A
Ruckpaul
K
K
D000595:Amino Acid Sequence; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003577:Cytochrome P-450 Enzyme System; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D007202:Indicators and Reagents; D007527:Isoenzymes; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification; D011817:Rabbits; D013861:Thiocyanates; D014326:Tropanes
D005452:Fluoresceins; D007202:Indicators and Reagents; D007527:Isoenzymes; D010455:Peptides; D013861:Thiocyanates; D014326:Tropanes; C008402:2-methoxy-5-nitrotropone; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D016650:Fluorescein-5-isothiocyanate