Probing the Role of the Hinge Segment of Cytochrome P450 Oxidoreductase in the Interaction with Cytochrome P450

NADPH-cytochrome P450 reductase (CPR) is the unique redox partner of microsomal cytochrome P450s (CYPs). CPR exists in a conformational equilibrium between open and closed conformations throughout its electron transfer (ET) function. Previously, we have shown that electrostatic and flexibility properties of the hinge segment of CPR are critical for ET. Three mutants of human CPR were studied (S243P, I245P and R246A) and combined with representative human drug-metabolizing CYPs (isoforms 1A2, 2A6 and 3A4). To probe the effect of these hinge mutations different experimental approaches were employed: CYP bioactivation capacity of pre-carcinogens, enzyme kinetic analysis, and effect of the ionic strength and cytochrome b5 (CYB5) on CYP activity. The hinge mutations influenced the bioactivation of pre-carcinogens, which seemed CYP isoform and substrate dependent. The deviations of Michaelis-Menten kinetic parameters uncovered tend to confirm this discrepancy, which was confirmed by CYP and hinge mutant specific salt/activity profiles. CPR/CYB5 competition experiments indicated a less important role of affinity in CPR/CYP interaction. Overall, our data suggest that the highly flexible hinge of CPR is responsible for the existence of a conformational aggregate of different open CPR conformers enabling ET-interaction with structural varied redox partners.

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The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b5

Archives of Biochemistry and Biophysics ◽

10.1016/j.abb.2010.10.023 ◽

2011 ◽

Vol 507 (1) ◽

pp. 144-153 ◽

Cited By ~ 86

Author(s):

Sang-Choul Im ◽

Lucy Waskell

Keyword(s):

Cytochrome P450 ◽

Cytochrome B5 ◽

Cytochrome P450 Reductase ◽

Microsomal Cytochrome ◽

P450 Reductase ◽

Redox Partners ◽

Cytochrome P450 2B4 ◽

Microsomal Cytochrome P450

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Reconstitution of the Enzymatic Activities of Cytochrome P450s Using Recombinant Flavocytochromes Containing Rat Cytochrome b5 Fused to NADPH–Cytochrome P450 Reductase with Various Membrane-Binding Segments

Archives of Biochemistry and Biophysics ◽

10.1006/abbi.2001.2372 ◽

2001 ◽

Vol 390 (2) ◽

pp. 215-221 ◽

Cited By ~ 15

Author(s):

Andrei A. Gilep ◽

Oleg L. Guryev ◽

Sergey A. Usanov ◽

Ronald W. Estabrook

Keyword(s):

Cytochrome P450 ◽

Cytochrome B5 ◽

Membrane Binding ◽

Cytochrome P450s ◽

Enzymatic Activities ◽

Cytochrome P450 Reductase ◽

Nadph Cytochrome ◽

P450 Reductase ◽

Nadph Cytochrome P450 Reductase

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Morphological adrenarche in rhesus macaques: development of the zona reticularis is concurrent with fetal zone regression in the early neonatal period

Journal of Endocrinology ◽

10.1677/joe-08-0337 ◽

2008 ◽

Vol 199 (3) ◽

pp. 367-378 ◽

Cited By ~ 26

Author(s):

Ann D Nguyen ◽

Samantha M Mapes ◽

C Jo Corbin ◽

Alan J Conley

Keyword(s):

Cytochrome P450 ◽

Rhesus Macaques ◽

Expression Profiles ◽

Cytochrome B5 ◽

Zona Reticularis ◽

Redox Partner ◽

Cytochrome P450 Oxidoreductase ◽

Dense Band ◽

Cytochrome P450 Family ◽

Steroid Dehydrogenase

Human adrenarche is associated with the establishment of a functional zona reticularis (ZR) and increasing secretion of dehydroepiandrosterone (DHEA) in sulfated form (DS). Like most non-human primates, rhesus macaques are not believed to undergo adrenarche, though they clearly establish a functional ZR after birth. However, the origins of the rhesus ZR are not well defined. Therefore, we investigated the zonal development, steroidogenic enzyme expression and morphology of rhesus adrenals from 1 day to 14 months of age. Immunohistochemistry was conducted to determine expression profiles of the steroidogenic enzymes 17α-hydroxylase/17,20-lyase cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1), cytochrome P450, family 21, subfamily A, polypeptide 2 (CYP21A2), hydroxy-Δ-5-steroid dehydrogenase, 3β- and steroid Δ-isomerase 2 (HSD3B2), the redox partner NADPH-cytochrome P450 oxidoreductase (CPR), as well as the accessory protein cytochrome b5 (b5), a marker of the primate ZR. The rhesus ZR is mature by 3 months of age based on differentiation of the innermost zone that lacks HSD3B2, but exhibits increased b5 expression during this period. Further, the ZR develops in neonates from a previously described dense band of cells which we show expresses b5, CYP17A1, CPR, and CYP21A2 throughout maturation. The fetal zone (FZ) is distinguished from the ZR by its lack of CYP21A2, and ZR development proceeded as the FZ regressed with two important implications: neither FZ regression nor ZR maturation can be monitored independently by circulating adrenal androgens, and these events must be induced by different factors in rhesus, and likely humans. Collectively these data demonstrate that ZR development begins before birth in the rhesus, proceeding concomitantly with FZ regression post-natally, suggesting that rhesus experiences morphological adrenarche during the first three months of life.

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