An Additional Electrostatic Interaction between Adrenodoxin and P450c27 (CYP27A1) Results in Tighter Binding Than between Adrenodoxin and P450scc (CYP11A1)

Irina A Pikuleva,Carolyn Cao,Michael R Waterman

doi:10.1074/jbc.274.4.2045

Abstract

Mitochondrial cytochrome P450c27 (product of the CYP27A1 gene) is found to have significantly higher affinity for the common redox partner adrenodoxin than another mitochondrial P450, P450scc (product of the CYP11A1 gene). To investigate the basis of the approximately 30-fold difference in adrenodoxin binding, two sets of P450c27 mutants were generated, expressed in Escherichia coli, and purified. Mutations of one set were within the putative adrenodoxin-binding site containing conserved lysine residues also crucial in P450scc for binding adrenodoxin. The second set included mutations within a sequence aligning with the "meander region" of P450BM-3 proposed to be a site of redox-partner interactions in P450s (Hasemann, C. A., Kurumbail, R. G., Boddupalli, S. S., Peterson, J. A., and Deisenhofer, J. (1995) Structure 3, 41-62). Mutation of the P450c27 conserved lysines (K354A and K358A) led to a approximately 20-fold increase in apparent Ks for adrenodoxin, confirming that these two positively charged residues conserved in mitochondrial P450s are important for adrenodoxin binding. Mutation of Arg-418, conserved in the CYP27A1 family, to serine also decreased the affinity for adrenodoxin approximately 20-fold. This residue is predicted to be located in the meander region. A triple K354A/K358A/R418S mutation profoundly reduced adrenodoxin binding. Thus, in contrast to P450scc, where mutation of the two conserved positively charged residues results in virtually complete inhibition of adrenodoxin binding, in P450c27 there are three of such residues (Lys-354, Lys-358, and Arg-418) important for adrenodoxin interaction.

Highlights

Proteins found in bacteria, yeast, fungi, nematodes, plants, insects, fish, and mammals [1]
Details of the interactions of different mitochondrial P450s with their common redox partner ferredoxin are derived from chemical modification and site-directed mutagenesis studies using bovine P450scc
In the present study it is confirmed with a second mitochondrial P450, human P450c27, that these conserved lysine residues are important for Adx binding

Summary

The abbreviations used are

P450, cytochrome P450; P450scc, cholesterol side chain cleavage cytochrome P450, product of the CYP11A1 gene; P450c27, cytochrome P450 catalyzing the multiple oxidation reactions at C-27 of steroids, product of the CYP27A1 gene; P450cam, cytochrome catalyzing the stereospecific hydroxylation of the camphor, product of the CYP101 gene; P450terp, cytochrome catalyzing the oxidation of the ␣-terpinol, product of the CYP108 gene; P450BM-3, cytochrome catalyzing the oxidation of a variety of fatty acids, product of the CYP102 gene; P45011␤, cytochrome P450 catalyzing the 11␤-hydroxylation of 11-deoxycortisol to cortisol and the production of aldosterone, product of the CYP11B1 gene [1]; Adx, adrenodoxin; Adr, adrenodoxin reductase; MALDI-TOF MS, matrix-assisted laser desorption ionization time of flight mass spectrometry. Proteins found in bacteria, yeast, fungi, nematodes, plants, insects, fish, and mammals [1] These monooxygenases metabolize different endogenous and exogenous substrates including steroids, fatty acids, drugs, and xenobiotics. By site-directed mutagenesis, lysine residues at positions 338 and 3422 (according to numbering of the mature protein) in P450scc were found to play crucial roles in binding bovine Adx [4]. The importance of these residues for Adx binding was implicated initially by chemical modification studies [5, 6]. The much tighter binding of P450c27 to Adx due to the presence of the additional positively charged residue may be important because of the low levels of ubiquitously expressed P450c27 and Adx relative to much higher levels of P450scc and Adx in steroidogenic mitochondria

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION