Functional interactions of adrenodoxin with several human mitochondrial cytochrome P450 enzymes

https://doi.org/10.1016/j.abb.2020.108596Get rights and content

Highlights

  • Human and bovine adrenodoxin behaved similarly in supporting human mitochondrial cytochrome P450 reactions.

  • Binding constants were estimated for adrenodoxin (Adx) and P450s and NADPH-Adx reductase using microscale thermophoresis.

  • A concentration of 10 µM adrenodoxin was optimal for all reactions examined using sub-µM concentrations of P450s.

Abstract

Seven of the 57 human cytochrome P450 (P450) enzymes are mitochondrial and carry out important reactions with steroids and vitamins A and D. These seven P450s utilize an electron transport chain that includes NADPH, NADPH-adrenodoxin reductase (AdR), and adrenodoxin (Adx) instead of the diflavin NADPH-P450 reductase (POR) used by the other P450s in the endoplasmic reticulum. Although numerous studies have been published involving mitochondrial P450 systems, the experimental conditions vary considerably. We compared human Adx and bovine Adx, a commonly used component, and found very similar catalytic activities in reactions catalyzed by human P450s 11B2, 27A1, and 27C1. Binding constants of 6–200 nM were estimated for Adx binding to these P450s using microscale thermophoresis. All P450 catalytic reactions were saturated at 10 μM Adx, and higher concentrations were not inhibitory up to at least 50 μM. Collectively these studies demonstrate the tight binding of Adx (both human and bovine) to AdR and to several mitochondrial P450s and provide guidance for optimization of Adx-dependent P450 reactions.

Introduction

The cytochrome P450 (P450 or CYP) family of heme monooxygenases has been of continued interest since the discovery of the enzyme more than 50 years ago [1,2]. With the sequencing of the human genome, the number of CYP genes was determined to be 57, and these enzymes play various roles in the body [3]. Most frequently they catalyze the insertion of a single atom from molecular oxygen into a C–H bond, using electrons supplied by NADPH, although they can perform reactions as wide-ranging as epoxidation, desaturation, C–C bond cleavage, and ring expansion [4,5]. In the liver, they are responsible for the activation and detoxication of xenobiotics, while in the adrenals, gonads, and placenta they catalyze key reactions in steroid hormone biosynthetic pathways [6]. P450 enzymes that metabolize exogenous chemicals, such as P450s 2C9 and 3A4, are generally localized to the endoplasmic reticulum and are dependent on the flavoprotein NADPH-P450 reductase (POR) for the transfer of electrons to support mixed-function oxidation. P450s in the mitochondria rely instead on a two-component electron transfer system comprised of a [2Fe–2S] ferredoxin, adrenodoxin (Adx), and an FAD-dependent ferredoxin reductase, NADPH-adrenodoxin reductase (AdR). Deficiencies in any of the mitochondrial P450s can cause disease states in humans involving not only steroidogenesis but also vitamin D homeostasis [[6], [7], [8]]. Of the six P450 enzymes involved in steroid hormone biosynthesis [3,6], three (P450s 11A1, 11B1, and 11B2) are mitochondrial P450s that use the Adx pathway. The remaining mitochondrial P450s also catalyze reactions in the bile acid pathway (P450 27A1 performs sterol 27-hydroxylation), vitamin D3 hydroxylation in the kidney (P450s 24A1 and 27B1), and retinoid desaturation in the skin (P450 27C1).

Because transfer of electrons to the P450 heme is mediated by the interaction of AdR, Adx, and the P450 itself, there has been significant interest over the years regarding the affinity and thermodynamics of the interactions between these components. The question of what complexes these form and how the formation affects the rate and specificity of electron transfer has been of interest since the first amino sequence determination of bovine Adx [9] and subsequent cloning and expression in Escherichia coli [10]. The first crystal structure of Adx was suggestive of dimerization [11]. More recently, Ivanov and associates used surface plasmon resonance (SPR) to estimate Kd values for POR and Adx interactions with 12 different P450s [12]. The effect of substrate binding on the P450-Adx complex has been examined recently both by SPR [13] and solution NMR [14,15]. Solution NMR indicated that substrate binding altered the interaction of microsomal P450 17A1 with another auxiliary protein, cytochrome b5 [16], which is consistent with more recent SPR studies [13]. In contrast, no effect of substrate was observed on P450/POR and P450/Adx binding with microsomal P450 17A1, P450 21A2, and P450 2C19 by SPR [13], while solution NMR showed that the mitochondrial P450 24A1/Adx interaction was sensitive to the presence of specific substrates [15]. These studies, taken together, indicate that Adx may play a role beyond simple delivery of electrons to P450s and, as a corollary, may additionally affect substrate binding and P450 conformation.

Studies of mitochondrial P450-catalyzed reactions have used a variety of experimental conditions in the interactions with Adx. Many of the early studies with Adx were done with the bovine protein, particularly prior to heterologous expression [[17], [18], [19], [20], [21]]. Taking P450 11B2 as an example, both (recombinant) human [[22], [23], [24]] and bovine [12,25,26] sources of Adx have been commonly used in catalytic studies. The concentrations of Adx and AdR used are generally described with regard to the concentration of P450 in a simple molar ratio. For typical steady-state enzyme kinetic studies, the Adx:P450 ratio has varied from 8:1 [24] to 60:1 [23] with Adx concentrations varying from 1 μM [25] to 30 μM [26].

In this work, we sought to understand the effects of these experimental variables in studies of mitochondrial P450s with Adx. Specifically, we examined the effects of different sources of Adx and concentrations on enzyme reaction parameters. Thermodynamic binding constants (Kd) for Adx and redox enzymes were estimated utilizing microscale thermophoresis (MST), again comparing the two Adx sources (bovine and human). The two Adx forms interact with the P450s differently, but the effect was found to be rather negligible for most enzyme kinetic studies. These results suggest optimized conditions for the study of interactions between Adx with mitochondrial P450s.

Section snippets

Chemicals

All chemical reagents were purchased from MilliporeSigma (Burlington, MA) or Thermo Fisher Scientific (Waltham, MA) and used without further purification unless otherwise noted.

Recombinant proteins

Bovine Adx and AdR [27], human P450 11B2 [25], human P450 27A1 [28], and human P450 27C1 [29] were all expressed in E. coli and purified as previously described. The plasmid containing human Adx (pLW01) was a gift from Prof. R. Auchus (U. Michigan) [22], and the protein was expressed and purified according to a similar

Bovine AdR

The Adx concentration-dependence of the catalytic activity of bovine AdR was tested with both human and bovine Adx. Reaction solutions were prepared in the wells of a polystyrene 96-well microplate (Greiner Bio-One North America, Monroe, NC). A 200 μL solution was prepared in each well including 0.015 μM AdR, 100 μM horse heart cytochrome c, 100 mM potassium phosphate buffer (pH 7.4), and various concentrations of either human or bovine Adx varying from 0.1 to 150 μM. NADPH (1.1 mM) was added

Enzyme catalytic activity dependence on Adx concentration

Steady-state kinetic measurements were made with three human mitochondrial P450 enzymes— P450s 11B2, 27A1, and 27C1—to compare the effectiveness of human and bovine Adx to act as redox mediators with human P450s. In each case the concentration of the P450 enzyme, the initial substrate concentration, and the concentration of bovine AdR were fixed. The concentration of the Adx was varied to examine the concentration-dependence of the observed P450 catalytic rate. Additional experiments to test

Discussion

The purpose of this study was to understand how variations in studies of P450- and Adx-catalyzed oxidations of steroids and vitamins affect the results. These variations include the species form of Adx used and the concentration of Adx used. The first issue is whether using bovine or human Adx as the electron carrier in P450 reactions changes the observed reaction kinetics. The primary sequences of the two proteins are very similar (76% identity), with most of the differences in the N-terminal

Conclusions

This goal of this work was to determine the effects of several experimental variables on reactions catalyzed by mitochondrial P450 enzymes, specifically understanding the effects of redox partner source and redox partner concentration. To achieve optimal enzyme catalysis, the Adx form utilized can be checked for optimal activity, but both bovine and human Adx led to rates within a factor of two. Regardless of the species origin of Adx, the concentration of Adx used in these studies must be

Acknowledgements

This work was supported by National Institutes of Health grants R01 GM118122 (F.P.G.), T32 ES007028 (F. P. G., M. J. R., S. M. G.), and F31 AR077386 (S. M. G.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors declare that they have no conflicts of interest with the contents of this article. The authors thank Ethan Harris for his assistance in preparing the AdR-cytochrome c enzyme

References (37)

Cited by (7)

  • Redox partner adrenodoxin alters cytochrome P450 11B1 ligand binding and inhibition

    2022, Journal of Inorganic Biochemistry
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    However, it is likely that adrenodoxin interactions and effects vary between P450 enzymes. Kinetic studies revealed that adrenodoxin mutations differentially affected the apparent Km for CYP11A1 and CYP11B1 [25] and catalytic rates depend differently on adrenodoxin concentration among P450 enzymes [13]. Additionally, the effect of the adrenodoxin oligomeric state on the allosteric effect is unknown and potentially additionally confounding.

  • Binding of cytochrome P450 27C1, a retinoid desaturase, to its accessory protein adrenodoxin

    2021, Archives of Biochemistry and Biophysics
    Citation Excerpt :

    P450 27C1 is an all-trans retinoid desaturase expressed in the skin [3,33] and is the only human mitochondrial P450 for which there is no structural information describing the interaction with Adx. Our group has previously measured the rate of P450 27C1 reduction by Adx (3.6 min−1 with substrate present) and the estimated binding affinity between the two proteins (220 nM) utilizing microscale thermophoresis (MST) [33,34], but additional details about the complex are not known. In this work, we characterized some aspects of the nature of the P450 27C1-Adx complex and investigated potential allosteric effects of Adx binding to P450 27C1.

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1

Co-first author.

2

Current address: Royal Society of Chemistry, RSC Publishing, Thomas Graham House (290), Science Park, Milton Road, Cambridge, CB4 0WF, United Kingdom.

3

Current address: Department of Chemistry and Fermentation Sciences, Appalachian State University, Boone, NC 28608, United States.

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