Chemical characterization of protein-protein interactions between cytochrome P-450 and cytochrome b5.

P P Tamburini,R E White,J B Schenkman

doi:10.1016/s0021-9258(18)89223-7

Abstract

Native cytochrome b5 interacts with either RLM5 or LM2 to form tight equimolar complexes (Kd = 250 and 540 nM, respectively) in which the content of high spin cytochrome P-450 was substantially increased. Cytochrome b5 caused 3- and 7-fold increases in the binding affinities of RLM5 and LM2 for benzphetamine, respectively, and benzphetamine decreased the apparent Kd for cytochrome b5 binding. Upon formation of the ternary complex between cytochromes P-450, b5, and benzphetamine the percentage of cytochrome P-450 in the high spin state was increased from 28 to 74 (RLM5) and from 9 to 85 (LM2). Cytochrome b5 caused 13- and 7-fold increases in the rate of RLM5- and LM2-dependent p-nitroanisole demethylation, respectively. Amino-modified (ethyl acetimidate or acetic anhydride) cytochrome b5 produced results similar to those obtained above with native cytochrome b5. In contrast, modification of as few as 5 mol of carboxyl groups/mol of amidinated cytochrome b5 resulted in both a substantial loss of the spectrally observed interactions with either cytochrome P-450 LM2 or cytochrome P-450 RLM5, and in a loss of the cytochrome b5-mediated stimulation of p-nitroanisole demethylation catalyzed by either monooxygenase. In further studies, native and fully acetylated cytochromes b5 reoxidized carbonmonoxy ferrous LM2 at least 20 times faster than amidinated, carboxyl-modified cytochrome b5 derivatives. In contrast, amidination, or acetylation of amino groups, or amidination of amino groups plus methylamidination of the carboxyl groups did not appreciably slow the rate of reduction of the cytochrome b5 by NADPH-cytochrome P-450 reductase. Collectively, the results provide strong evidence for an essential role of cytochrome b5 carboxyl groups in functional interactions with RLM5 and LM2.

Highlights

Chrome P-450 (Imai and Sato,1977; Ingelman-Sundberg and modification of as few as 5 mol ofcarboxyl groups/mol Johansson, 1980; Bonfils et al, 1981), or improved coupling of amidinated cytochrome bs resulted in both a sub- between oxyferrous cytochrome P-450 and NADPH-cytostantial loss of the spectrally observed interactions with either cytochrome P-450 LM2or cytochrome P460 RLMs, and ina loss of the cytochrome b6-mediated stimulation of p-nitroanisole demethylation catalyzed by either monooxygenase
In order to investigate this and Coon, 1968).This system functions in the oxidation of a possibility we have studied the effect of selective chemical diversity of chemical structures, including xenobiotics (Lu et modification of both amino and carboxyl groups of cytoal., 1973),fatty acids (Lu andCoon, 1968;Gibson et al, 1980), chrome bs upon both the thermodynamic and catalytic intersteroids
The corresponding spectral interactionsbetween cytochrome P-450 LM2 and cytochrome b5 are shown in Fig. 2 for comparison

Summary

MATERIALS AND METHODS

Eluting fractions displaying an A,12/Am > 2.24 were pooled (800 nmol) and applied to a column of Sephadex G-25 (1.4 X 27 cm) previously equilibrated with 50 mM sodium phosphate buffer, pH 7.25, followed bydialysis uersus two successive 2-liter volumes of the same buffer. Solid methylamine hydrochloride and "Cmethylamine HC1 (46 mCi/mmol) were added to amidinated cytochrome bs (140 HM)in 10 mM sodium phosphate buffer, pH 7.0, to attain a final concentration of 0.5 M and specific activity of approximately 110 cpm/nmol. Cytochrome b6 or its modified derivatives were present at a final concentration of 0.6 p ~ A.fter establishinga base-line absorbance with time for 5 min at 25"C (versus 30 pg ml"of DLPC in 50 m M sodium phosphate, pH 7.25, 25% glycerol), the reaction was initiated by the addition of NADPH to a 1 mM final concentration.

RESULTS

P-450 Benzphetamine

DISCUSSION