Adrenal steroid hydroxylases. Oxidation- reduction properties of adrenal iron-sulfur protein (adrenodoxin)

Mitochondrial cytochromes P450 (P450s) are responsible for important metabolic reactions, including steps involved in steroid and vitamin D metabolism. The mitochondrial P450 24A1 (CYP24A1) is responsible for deactivation of the bioactive form of vitamin D, 1,25(OH) 2 D3. Its function relies on formation of a P450-redox partner complex with the ferredoxin and electron donor adrenodoxin (Adx). However, very little is known about how the Adx-CYP24A1 complex forms. In this study, we report the results of solution NMR in which we monitor isotopically labeled full-length Adx as it binds CYP24A1 in complex with the P450 inhibitor clotrimazole. The NMR titration data suggested a mode for P450-Adx interactions in which formation of the complex relies on contributions from multiple recognition sites on the Adx core domain, some of which have not previously been reported. To evaluate differences among CYP24A1-Adx complexes from different mammalian species and displaying distinct regioselectivity for 1,25(OH) 2 D3, all bound spectra were acquired in parallel for human (carbon-23 and -24 hydroxylase), rat (carbon-24 hydroxylase), and opossum (carbon-23 hydroxylase) CYP24A1 isoforms. Binding data from a series of single and double charge-neutralizing substitutions of Adx confirmed that species-specific CYP24A1 isoforms differ in binding to Adx, providing evidence that variations in redox partner interactions correlate with P450 regioselectivity. In summary, these findings reveal that CYP24A1-Adx interactions rely on several recognition sites and that variations in CYP24A1 isoforms modulate formation of the complex, thus providing insight into the variable and complex nature of mitochondrial P450-Adx interactions.Mitochondrial cytochromes P450 (P450) are responsible for a host of biological reactions, including steps necessary in steroid and vitamin D metabolism. Similar to microsomal P450s, their function requires the transfer of two electrons, delivered sequentially, in order to generate the active oxidizing species necessary for completion of one cycle of P450 catalysis (1). However, in contrast to microsomal P450 enzymes, for which reduction of P450 can be achieved by binding either a P450 oxidoreductase or, in some cases, the small heme protein cytochrome b 5 , mitochondrial enzymes rely entirely on formation of a transient complex with the soluble ferredoxin protein, Adrenodoxin (Adx). Adx folds into a compact structure consisting of three short α-helices and five anti-parallel β strands, with a [2Fe-2S] cluster coordinated near solvent accessible surfaces between helix-1 and helix-3 (2,3). Changes in the C-terminal region of bovine Adx have been shown to participate in mediating a monomer to dimer transition in response to reduction of the [2Fe-2S] cluster (2), thereby suggesting that dimers of Adx are functionally relevant.While the prevailing evidence suggests that complex formation between Adx with P450 relies on salt bridge interactions between the side chains of acidic residues on helix-3 (Asp-72 - Cytoch...

Section: Methodsmentioning

confidence: 99%

The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

Estrada

2018

“…These cultures were used to inoculate 4 liters of a main culture containing ampicillin. Isopropyl-1-thio-␤-D-galactopyranoside was added to induce heterologous protein production, and afterward cultures were grown at 37°C for 16 h. Recombinant Adx was purified after sonification as described, and the final concentration of Adx was determined using ⑀ 414 ϭ 9.8 mM Ϫ1 cm Ϫ1 (27). The purity of the Adx preparation was estimated by determining the relative absorbance of the protein at 414 nm and 273 nm, Q value (A 414 /A 273 ).…”

Section: Methodsmentioning

confidence: 99%

Stripping Down the Mitochondrial Cholesterol Hydroxylase System, a Kinetics Study

Schiffler

Zöllner

Bernhardt

2004

The origin of steroid hormones in mammals is cholesterol that is metabolized by the mitochondrial CYP11A1 system. The cytochrome P450 is fed with reduction equivalents via a small electron transfer chain consisting of NADPH, adrenodoxin reductase, and adrenodoxin. Though the redox behavior of the individual protein components has been studied previously, the kinetics of the system in its entirety has not yet been analyzed. In this study we combine surface plasmon resonance experiments to determine the binding constants for the different pairs of redox partners with measurements of the pre-steady-state kinetics of the different reaction steps of this system and steady-state kinetics. We could correlate the individual protein-protein interactions with the effect of distinct reductionoxidation steps on the overall catalytic activity of the CYP11A1 system. For the first time, we were able to follow the reduction of each of the protein components of this system within one measurement when we mixed all oxidized protein components with NADPH. These measurements allowed the determination of the individual apparent rate constants for the reduction of all three proteins involved. In addition, variation of the ionic strength in these experiments revealed different optimum salt concentrations for the reduction of adrenodoxin reductase and adrenodoxin, respectively, and unraveled dramatically changing reduction rates of CYP11A1 by adrenodoxin.The mitochondrial steroid hydroxylase CYP11A1 1 belongs to the superfamily of cytochrome P450 enzymes. It catalyzes the rate-limiting step in the biogenesis of steroid hormones in adrenal mitochondria (1, 2). In this reaction, the side chain of the substrate cholesterol gets cleaved off to yield pregnenolone and isocaproic aldehyde at the expense of three molecules of NADPH and three molecules of oxygen (3). The reduction equivalents, provided by NADPH, are transferred to a FAD containing adrenodoxin reductase (AdR), which transfers electrons to the one electron carrier adrenodoxin (Adx), the ferredoxin of the adrenal gland (4). The [2Fe-2S] cluster-containing Adx subsequently transfers electrons, one at a time, to the heme iron of CYP11A1. These electrons are necessary for the reductive activation of oxygen and finally to hydroxylate the substrate. For a complete conversion of cholesterol to pregnenolone, six electrons have to be supplied.About 40 years ago the group of Kimura (5-8) started to characterize single components of the system followed by a multitude of investigations concerning the adrenal steroid hydroxylase system and its different components by various groups (9 -18). During the last decade much work has been done to get a better understanding of the kinetics of this system (4, 19 -23). Nevertheless, the nature of the rate-limiting step in this system is still a matter of controversial discussion and remains to be elucidated.For the first time we were able to follow the reduction of all three protein components of the CYP11A1 system within one measurement using rapid mix...

“…Recombinant adrenodoxin was purified after enzymatic cell lysis as described previously, and the final concentration of adrenodoxin was determined using ⑀ 414 ϭ 9.8 mM Ϫ1 cm Ϫ1 (31). The purity of the adrenodoxin preparation was estimated by determining the Q-value (A 414 /A 273 ).…”

Section: Methodsmentioning

confidence: 99%

The Interaction of Bovine Adrenodoxin with CYP11A1 (Cytochrome P450scc) and CYP11B1 (Cytochrome P45011β)

Schiffler

Kiefer

Wilken

et al. 2001

The kinetics of protein-protein interaction and heme reduction between adrenodoxin wild type as well as eight mutants and the cytochromes P450 CYP11A1 and CYP11B1 was studied in detail. Rate constants for the formation of the reduced CYP11A1⅐CO and CYP11B1⅐CO complexes by wild type adrenodoxin, the adrenodoxin mutants Adx-(4 -108), Adx-(4 -114), T54S, T54A, and S112W, and the double mutants Y82F/S112W, Y82L/ S112W, and Y82S/S112W (the last four mutants are ⌬113-128) are presented. The rate constants observed differ by a factor of up to 10 among the respective adrenodoxin mutants for CYP11A1 but not for CYP11B1. According to their apparent rate constants for CYP11A1, the adrenodoxin mutants can be grouped into a slow (wild type, T54A, and T54S) and a fast group (all the other mutants). The adrenodoxin mutants forming the most stable complexes with CYP11A1 show the fastest rates of reduction and the highest rate constants for cholesterol to pregnenolone conversion. This strong correlation suggests that C-terminal truncation of adrenodoxin in combination with the introduction of a C-terminal tryptophan residue enables a modified protein-protein interaction rendering the system almost as effective as the bacterial putidaredoxin/CYP101 system. Such a variation of the adrenodoxin structure resulted in a mutant protein (S112W) showing a 100-fold increased efficiency in conversion of cholesterol to pregnenolone.Cytochromes P450 (CYP11A1, CYP11B1, and CYP11B2) 1 of the inner mitochondrial membrane catalyze various hydroxylation steps in the biosynthesis of steroid hormones (mineralocorticoids, glucocorticoids, and androgens) in vertebrates (1, 2). The electrons required for these hydroxylation reactions are provided by NADPH and are transferred to the cytochromes P450, which mediate the final oxygen activation, via a small electron transport chain. Comparable with the components of the soluble bacterial cytochrome P450 systems, such as P450 cam (CYP101) and P450 terp (CYP108), the proteins of this electron transport system are an FAD-containing NAD(P)H-dependent reductase and an iron-sulfur protein of the [2Fe-2S] ferredoxin type.Adrenodoxin, the ferredoxin of the adrenal gland (3), has been studied intensively by chemical modifications (4 -6) and later by a great number of site-directed mutagenesis studies (7-13). Recently crystal structures of a truncated form, Adx-(4 -108) (14), and of full-length Adx (15) have been solved. In contrast to the bacterial systems, the reductase and the cytochromes of the mitochondrial systems are membrane-associated or membrane-bound proteins, respectively (1, 16).The reduction kinetics of the soluble bacterial cytochrome P450 cam is well understood (17-20), whereas little is known about the kinetics of the membrane-bound mitochondrial cytochromes P450 (21-23). Nevertheless previous investigations point to considerable differences in the reaction mechanisms and in the regulation in these two types of cytochrome P450 systems.To get deeper insight into the mechanism causing these differenc...