Nanosecond fluorescence of tryptophans in cytochrome P-450scc (CYP11A1): effect of substrate binding.

Fluorescence of eight tryptophan residues in cytochrome P-450scc with bound endogenous cholesterol could be fitted with a two component model: a single exponential and a "top-hat" distribution of lifetimes as the second component. The short-lived component (tau 1 about 700 ps) does not change significantly upon binding of substrate (22R-hydroxycholesterol). The parameters of the long-lived component (central lifetime tau m about 3.4 ns) change upon binding of carbon monoxide and substrate. 22R-hydroxycholesterol binding broadens the distribution of the long-lived component; that is the heterogeneity of the Trp environment is increased when this substrate displaces the endogenous cholesterol.     

Interaction of CYP11B1 (cytochrome P-45011 beta) with CYP11A1 (cytochrome P-450scc) in COS-1 cells.

The interactions of CYP11B1 (cytochrome P-45011beta), CYP11B2 (cytochrome P-450aldo) and CYP11A1 (cytochrome P-450scc) were investigated by cotransfection of their cDNA into COS-1 cells. The effect of CYP11A1 on CYP11B isozymes was examined by studying the conversion of 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and aldosterone. It was shown that when human or bovine CYP11B1 and CYP11A1 were cotransfected they competed for the reducing equivalents from the limiting source contained in COS-1 cells; this resulted in a decrease of the CYP11B activities without changes in the product formation patterns. The competition of human CYP11A1 with human CYP11B1 and CYP11B2 could be diminished with excess expression of bovine adrenodoxin. However, the coexpression of bovine CYP11B1 and CYP11A1 in the presence of adrenodoxin resulted in a stimulation of 11beta-hydroxylation activity of CYP11B1 and in a decrease of the 18-hydroxycorticosterone and aldosterone formation. These results suggest that the interactions of CYP11A1 with CYP11B1 and CYP11B2 do not have an identical regulatory function in human and in bovine adrenal tissue.     

Probing the interaction of bovine cytochrome P450scc (CYP11A1) with adrenodoxin: evaluating site-directed mutations by molecular modeling

The present study was undertaken to evaluate the role of positively charged amino acid residues proposed to reside on the proximal surface of bovine cytochrome P450 cholesterol side chain cleavage (P450scc, CYP11A1) and to determine which residues may be involved in protein-protein interactions with the electron carrier adrenodoxin (Adx). In previous studies, nine different lysine residues were identified by chemical and immunological cross-linking experiments as potentially interacting with Adx, while in the present study, two arginine residues have been identified from sequence alignments. From these 11 residues, 13 different P450scc mutants were made of which only seven were able to be expressed and characterized. Each of the seven mutants were evaluated for their ability to bind Adx, to be reduced, and for their enzymatic activity. Among these, K403Q and K405Q showed a consistent decrease in Adx binding, the ability to be reduced by Adx, and enzymatic activity, with K405Q being affected to a much greater extent. More dramatic was the complete loss of Adx binding by R426Q, while still retaining its ability to be chemically reduced and bind carbon monoxide. Independently, a homology model of P450scc was constructed and docked with the structure of Adx. Four potential sites of interaction were identified: P450scc:K403 with Adx:D76, P450scc:K405 with Adx:D72; P450scc:R426 with Adx:E73, and P450scc:K267 with Adx:E47. Thus, the biochemical and molecular modeling studies together support the hypothesis that K267, K403, K405, and R426 participate in the electrostatic interaction of P450scc with Adx.     

The interaction of bovine adrenodoxin with CYP11A1 (cytochrome P450scc) and CYP11B1 (cytochrome P45011beta ). Acceleration of reduction and substrate conversion by site-directed mutagenesis of adrenodoxin.

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 CYP11A1.CO and CYP11B1.CO 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 Delta113-128) 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.     

Phosphorylation of bovine adrenodoxin by protein kinase CK2 affects the interaction with its redox partner cytochrome P450scc (CYP11A1)

Adrenodoxin (Adx), a [2Fe-2S] vertebrate-type ferredoxin, transfers electrons from the NADPH-dependent flavoprotein Adx reductase (AdR) to mitochondrial cytochrome P450 enzymes of the CYP11A and CYP11B families, which catalyze key reactions in steroid hormone biosynthesis. Adx is a known phosphoprotein, but the kinases that phosphorylate Adx have remained mostly obscure. The aim of this study was to identify previously unknown Adx phosphorylating kinases and to acquire a deeper insight into the functional consequences of such a modification. Here, we show for the first time that bovine Adx is a substrate of protein kinase CK2, whereas bovine CYP11A1, CYP11B1, and AdR are not phosphorylated by this kinase. CK2 phosphorylation of mature Adx requires the presence of both the catalytic alpha-subunit and the regulatory beta-subunit of CK2 and takes place exclusively at residue Thr-71, which is located within the redox partner interaction domain of the protein. We created two Adx mutants, Adx-T71E (imitating a phosphorylation) and Adx-T71V (which cannot be phosphorylated at this site), respectively, and investigated how these mutations affected the interaction of Adx with its redox partners. These data were supplemented with detailed spectroscopic and functional assays using the phosphorylated protein. All Adx species behaved like wild type (Adx-WT) with respect to their redox potential, iron-sulfur cluster symmetry, and overall backbone structure. Substrate conversion assays catalyzed by CYP11A1 showed an increase in product formation when Adx-T71E or CK2-phosphorylated Adx were used as electron carrier instead of Adx-WT, whereas the activity toward CYP11B1 was not altered using these Adx species. Additionally, Adx-T71E represents the only full-length Adx mutant which leads to an increase in CYP11A1 product formation. Therefore, characterizing this full-length mutant helps to improve our knowledge on the functional effects of phosphorylations on complex redox systems.     

Kinetics of vitamin D3 metabolism by cytochrome P450scc (CYP11A1) in phospholipid vesicles and cyclodextrin

Vitamin D3 can be hydroxylated sequentially by cytochrome P450scc (CYP11A1) producing 20-hydroxyvitamin D3, 20,23-dihydroxyvitamin D3 and 17,20,23-trihydroxyvitamin D3. The aim of this study was to characterize the ability of vitamin D3 to associate with phospholipid vesicles and to determine the kinetics of metabolism of vitamin D3 by P450scc in vesicles and in 2-hydroxypropyl-beta-cyclodextrin (cyclodextrin). Gel filtration of phospholipid vesicles showed that the vitamin D3 remained quantitatively associated with the phospholipid membrane. Vitamin D3 exchanged between vesicles at a rate 3.8-fold higher than for cholesterol exchange and was stimulated by N-62 StAR protein. The Km of P450scc for vitamin D3 in vesicles was 3.3 mol vitamin D3/mol phospholipid and the rate of conversion of vitamin D3 to 20-hydroxyvitamin D3 was first order with respect to the vitamin D3 concentration for the range of concentrations of vitamin D3 that could be incorporated into the vesicle membrane. 20-Hydroxyvitamin D3 was further hydroxylated by P450scc in vesicles, producing primarily 20,23-dihydroxyvitamin D3, with Km and kcat values 22- and 6-fold lower than those for vitamin D3, respectively. 20,23-dihydroxyvitamin D3 was converted to 17,20,23-trihydroxyvitamin D3 with even lower Km and kcat values. Vitamin D3 and cholesterol were metabolized with comparable efficiencies in cyclodextrin, but the Km for both showed a strong dependence on the cyclodextrin concentration, decreasing with decreasing cyclodextrin. This study shows that vitamin D3 quantitatively associates with phospholipid vesicles, can exchange between membranes, and can be hydroxylated by membrane-associated P450scc but with lower efficiency than for cholesterol hydroxylation. The kcat values for metabolism of vitamin D3 in vesicles and 0.45% cyclodextrin are similar, but the ability to solubilize vitamin D3 at a concentration higher than its Km makes the cyclodextrin system more efficient for producing the hydroxyvitamin D3 metabolites for further characterization.     

The F-G loop region of cytochrome P450scc (CYP11A1) interacts with the phospholipid membrane

Cytochrome P450scc (CYP11A1) is a protein attached to the inner surface of the inner mitochondrial membrane that uses cholesterol from the membrane phase as its substrate for the first step in steroid hormone synthesis. We investigated the mechanism by which CYP11A1 interacts with the membrane. Hydrophobicity profiles of CYP11A1 and two other mitochondrial cytochromes P450, plus a model structure of CYP11A1 using CYP2C5 as template, suggest that CYP11A1 has a monotopic association with the membrane which may involve the A' helix and the F-G loop. Deletion of the A' helix reduced the proportion of expressed CYP11A1 associated with the bacterial membrane fraction, indicating a role for the A' helix in membrane binding. However, introduction of a cysteine residue in this helix at position 24 (L24C) and subsequent labelling with the fluorescent probe N'-(7-nitrobenz-2-oxal,3-diazol-4-yl)ethylenediamine (NBD) failed to show a membrane localisation. Cysteine mutagenesis and fluorescent labelling of other residues appearing on the distal surface of the CYP11A1 model revealed that V212C and L219C have enhanced fluorescence and a blue shift following association of the mutant CYP11A1 with phospholipid vesicles. This indicates that these residues, which are located in the F-G loop, become localised to a more hydrophobic environment following membrane binding. Analysis of the quenching of tryptophan residues in CYP11A1 by acrylamide indicates that at least one and probably two tryptophans are involved in membrane binding. We conclude that CYP11A1 has a monotopic association with the membrane that is mediated, at least in part, by the F-G loop region.     

The effect of glycerol on cytochrome P450scc (CYP11A1) spin state,activity, and hydration

We examined the effects of glycerol, a stabilizing agent commonly used in cytochrome P450scc purification and analysis, on the spin state, catalytic activity, and molecular volume of the cytochrome. Glycerol induced a sigmoidal low-spin response. The binding of hydroxycholesterol reaction intermediates, but not cholesterol, increased the concentration of glycerol required for the spin transition to be 50% complete (K(1/2)). Glycerol weakened adrenodoxin binding to P450scc but had no effect on CO or 20alpha,22R-dihydroxycholesterol binding. Cytochrome P450scc activity was inhibited by glycerol with the K(1/2) for inhibition being substrate-dependent. The osmotic stress exerted by glycerol on P450scc resulted in decreases in P450scc molecular volume for both the transition to low spin state and the inhibition of activity. From this we determined that two dissociative water molecules are involved in the inhibition of activity with cholesterol as substrate and five or six dissociative waters are involved in the low-spin transition. The dehydration of P450scc by osmotic stress provides an explanation for the effects of glycerol on P450scc spin transition and activity.     

Placental cytochrome P450scc (CYP11A1): comparison of catalytic properties between conditions of limiting and saturating adrenodoxin reductase

The mitochondrial side-chain cleavage of cholesterol, catalysed by cytochrome P450scc, is rate-limiting in the synthesis of progesterone by the human placenta. Cytochrome P450scc activity is in turn limited by the concentration of adrenodoxin reductase (AR) in placental mitochondria. In order to better understand which components of the cholesterol side-chain cleavage system are important in the regulation of placental progesterone synthesis, we have examined their effects on P450scc activity with both saturating and limiting concentrations of AR. The present study reveals that decreasing the AR concentration causes a decrease in the K(m) of cytochrome P450scc for cholesterol, facilitating saturation of the enzyme with its substrate. Decreasing AR resulted in P450scc activity becoming less sensitive to changes in P450scc concentration. The adrenodoxin (Adx) concentration in mitochondria from term placentae is near-saturating for P450scc and under these conditions, we found that decreasing AR reduces the K(m) of P450scc for adrenodoxin. Increasing either the cholesterol or P450scc concentration increased the amount of AR required for P450scc to work at half its maximum velocity. A relatively small increase in AR can support considerably higher rates of side-chain cleavage activity when there is a coordinate increase in AR and P450scc concentrations. We conclude from this study that cholesterol is near-saturating for cytochrome P450scc activity in placental mitochondria due to the P450scc displaying a low K(m) for cholesterol resulting from the low and rate-limiting concentration of AR present. This study reveals that it is unlikely that cholesterol or adrenodoxin concentrations are important regulators of placental progesterone synthesis but AR or coordinate changes in AR and P450scc concentrations are likely to be important in its regulation.     

Adrenodoxin (Adx) and CYP11A1 (P450scc) induce apoptosis by the generation of reactive oxygen species in mitochondria

Mitochondrial cytochrome P450 systems are an indispensable component of mammalian steroid biosynthesis; they catalyze regio- and stereo-specific steroid hydroxylations and consist of three protein entities: adrenodoxin reductase (AdR), adrenodoxin (Adx), and a mitochondrial cytochrome P450 enzyme, e.g., CYP11A1 (P450 side chain cleavage, P450scc). It is known that the latter two are able to generate reactive oxygen species (ROS) in vitro . In this study, we investigated whether this ROS generation also occurs in vivo and, if so, whether it leads to the induction of apoptosis. We found that overexpression of either human or bovine Adx causes a significant loss of viability in 11 different cell lines. This loss of viability does not depend on the presence of the tumor suppressor protein p53. Transient overexpression of human Adx in HCT116 cells leads to ROS production, to a disruption of the mitochondrial transmembrane potential (DeltaPsi), to cytochrome c release from the mitochondria, and to caspase activation. In contrast, the effect of transient overexpression of human CYP11A1 on cell viability varies in different cell lines, with some being sensitive and others not. We conclude that mitochondrial cytochrome P450 systems are a source of mitochondrial ROS production and can play a role in the induction of mitochondrial apoptosis.     

Complex formation in vesicle-reconstituted mitochondrial cytochrome P450 systems (CYP11A1 and CYP11B1) as evidenced by rotational diffusion experiments using EPR and ST-EPR.

Rotational diffusion measurements using EPR and saturation transfer EPR were applied to analyze complex formation between the electron-transfer components of the mitochondrial steroid-hydroxylating cytochrome P450 systems (CYP11A1 and CYP11B1) in phosphatidylcholine/phosphatidylethanolamine/cardiolipin vesicles prepared by octyl glucoside dialysis/adsorption. Octyl glucoside reconstitution of P450SCC results in large vesicles, which have an advantage over small vesicles in that vesicle tumbling does not contribute to measured rotational diffusion rates. Immobilization of spin-labeled adrenodoxin by both P450SCC and adrenodoxin reductase indicates equimolar complexation between P450SCC and adrenodoxin as well as between adrenodoxin reductase and adrenodoxin. Combination of rotational diffusion and antibody cross-linking confirmed the complexation of adrenodoxin with P450SCC and for the first time provided direct evidence of a complex between P450SCC and P45011beta in the membrane. In contrast, no evidence was found for the existence of adrenodoxin reductase-P450SCC complexes or a ternary complex of all three proteins. Thus, these experiments confirm the shuttle mechanism of electron transfer to vesicle-reconstituted P450SCC and P45011beta.     

Substrate-binding region of cytochrome P-450scc (P-450 XIA1). Identification and primary structure of the cholesterol binding region in cytochrome P-450scc

Cytochrome P-450_scc (P-450 XIA1) from bovine adrenocortical mitochondria was investigated using a suicide substrate: [¹⁴C]methoxychlor. [¹⁴C]Methoxychlor irreversibly abolished the activity of the side-chain cleavage enzyme for cholesterol (P-450_scc) and the inactivation was prevented in the presence of cholesterol. The binding of [¹⁴C]methoxychlor and cytochrome P-450_scc occurred in a molar ratio of 1:1 and the cholesterol-induced difference spectrum of cytochrome P-450_scc was similar with the methoxychlor-induced difference spectrum. [¹⁴C]Methoxychlor-binding peptides were purified from tryptic-digested cytochrome P-450_scc modified with [¹⁴C]methoxychlor. Determination of the sequence of the amino-acid residues of a [¹⁴C]methoxychlor-binding peptide allowed identification of the peptide comprising the amino-terminal amino-acid residues 8 to 28.     

Comparative structural-functional characterization of recombinant and natural adrenodoxin. Interaction with cytochrome P450scc.

The conditions for heterologous expression of recombinant bovine adrenodoxin in E. coli have been optimized, thus reaching expression levels up to 12-14 micromoles per liter of culture medium. A highly efficient method for purification of this recombinant ferredoxin from the E. coli cells has been developed. The structural-functional properties of the highly purified recombinant protein have been characterized and compared to those of natural adrenodoxin purified from bovine adrenocortical mitochondria. In contrast to natural adrenodoxin, which is characterized by microheterogeneity, the recombinant adrenodoxin is homogeneous as judged by N- and C-terminal amino acid sequencing, and its sequence corresponds to the full-length mature form of adrenodoxin containing 128 amino acid residues. The interactions of the natural and recombinant adrenodoxins with cytochrome P450scc have been studied and compared with respect to: the efficiency of their enzymatic reduction of cytochrome P450scc in a reconstituted system; the ability of the immobilized adrenodoxins to bind cytochrome P450scc; the ability of the adrenodoxins to induce a spectral shift of cytochrome P450scc and to effect the average polarity of exposed tyrosines in the low-spin cytochrome P450scc. The recombinant adrenodoxin is functionally active and in the reduced state as well as at low ionic strength it displays higher affinity to cytochrome P450scc as compared to the natural bovine adrenocortical adrenodoxin. The possible role of the C-terminal sequence of the adrenodoxin molecule in its interaction with cytochrome P450scc as well as the advantages of using the recombinant protein instead of the natural one are discussed.     

Peripheral ligand-binding site in cytochrome P450 3A4 located with fluorescence resonance energy transfer (FRET)

The mechanisms of ligand binding and allostery in the major human drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) were explored with fluorescence resonance energy transfer (FRET) using a laser dye, fluorol-7GA (F7GA), as a model substrate. Incorporation into the enzyme of a thiol-reactive FRET probe, pyrene iodoacetamide, allowed us to monitor the binding by FRET from the pyrene donor to the F7GA acceptor. Cooperativity of the interactions detected by FRET indicates that the enzyme possesses at least two F7GA-binding sites that have different FRET efficiencies and are therefore widely separated. To probe spatial localization of these sites, we studied FRET in a series of mutants bearing pyrene iodoacetamide at different positions, and we measured the distances from each of the sites to the donor. Our results demonstrate the presence of a high affinity binding site at the enzyme periphery. Analysis of the set of measured distances complemented with molecular modeling and docking allowed us to pinpoint the most probable peripheral site. It is located in the vicinity of residues 217-220, similar to the position of the progesterone molecule bound at the distal surface of the CYP3A4 in a prior x-ray crystal structure. Peripheral binding of F7GA causes a substantial spin shift and serves as a prerequisite for the binding in the active site. This is the first indication of functionally important ligand binding outside of the active site in cytochromes P450. The findings strongly suggest that the mechanisms of CYP3A4 cooperativity involve a conformational transition triggered by an allosteric ligand.     

Distance between lysine 384 and heme of cytochrome P-450 LM2 (P-450 IIB4) studied by fluorescence energy transfer measurements

The distance between FITC-modified lysine 384 of cytochrome P-450 LM2 and the active site, heme, was estimated by fluorescence energy transfer measurements. To avoid differential labelling of P-450 LM2 for protection of the alpha-amino group from FITC modification, deconvolution of measured fluorescence decay curves using a double exponential model was performed. A value of 2.7 nm was obtained for the distance FITC (lysine 384) - heme. This distance is too large to account for a direct electron tunneling from prosthetic group to prosthetic group at this interaction site between reductase and P-450 LM2.     

Clay-bridged electron transfer between cytochrome p450(cam) and electrode

We demonstrate a very fast heterogeneous redox reaction of substrate-free cytochrome P450(cam) on a glassy carbon electrode modified with sodium montmorillonite. The linear relationship of the peak current in the cyclic voltammogram with the scan rate indicates a reversible one-electron transfer surface process. The electron transfer rate is in the range from 5 to 152 s(-1) with scan rates from 0.4 to 12 V/s, respectively. These values are comparable to rates reported for the natural electron transfer from putidaredoxin to P450(cam). The formal potential of adsorbed P450(cam) is -139 mV (vs NHE) and therefore positively shifted by 164 mV compared to the potential of substrate-free P450(cam) in solution. UV-VIS and FTIR spectra do not indicate an influence of the clay colloidal particles on the heme and the secondary structure of P450(cam) in solution. However, P450(cam) adsorbed on the surface of the clay-modified electrode may undergo partial dehydration resulting in the shift of the formal potential.     

Active site of bovine adrenocortical cytochrome P-450(11) beta studied by resonance Raman and electron paramagnetic resonance spectroscopies: distinction from cytochrome P-450scc

Cytochrome P-45011 beta was purified as the 11-deoxycorticosterone-bound form from bovine adrenocortical mitochondria and its active site was investigated by resonance Raman and EPR spectroscopies. Resonance Raman spectra of the purified sample revealed that the heme iron adopts the pure pentacoordinated ferric high-spin state on the basis of the nu 10 (1629cm-1) and nu 3 (1490 cm-1) mode frequencies, which are higher than those of the hexacoordinated ferric high-spin cytochrome P-450scc-substrate complexes. In the ferrous-CO state, a Fe2(+)-CO stretching mode was identified at 481.5 cm-1 on the basis of an isotopic substitution technique; this frequency is very close to that of cytochrome P-450scc in the cholesterol-complexed state (483 cm-1). The EPR spectra of the purified sample at 4.2 K showed ferric high-spin signals (at g = 7.98, 3.65, and 1.71) that were clearly distinct from the cytochrome P-450scc ferric high-spin signals (g = 8.06, 3.55, and 1.68) and confirmed previous assignments of ferric high-spin signals in adrenocortical mitochondria. The EPR spectra of the nitric oxide (NO) complex of ferrous cytochrome P-45011 beta showed EPR signals with rhombic symmetry (gx = 2.068, gz = 2.001, and gy = 1.961) very similar to those of the ferrous cytochrome P-450scc-NO complex in the presence of 22(S)-hydroxycholesterol and 20(R),22-(R)-dihydroxycholesterol at 77 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deletions in the loop surrounding the iron-sulfur cluster of adrenodoxin severely affect the interactions with its native redox partners adrenodoxin reductase and cytochrome P450(scc) (CYP11A1)

The redox active iron-sulfur center of bovine adrenodoxin is coordinated by four cysteine residues in positions 46, 52, 55 and 92 and is covered by a loop containing the residues Glu-47, Gly-48, Thr-49, Leu-50 and Ala-51. In plant-type [2Fe-2S] ferredoxins, the corresponding loop consists of only four amino acids. The loop is positioned at the surface of the proteins and forms a boundary separating the [2Fe-2S] cluster from solvent. In order to analyze the biological function of the five amino acids of the loop in adrenodoxin (Adx) for this electron transfer protein each residue was deleted by site-directed mutagenesis. The resulting five recombinant Adx variants show dramatic differences among each other regarding their spectroscopic characteristics and functional properties. The redox potential is affected differently depending on the position of the conducted deletion. In contrast, all mutations in the protein loop influence the binding to the redox partners adrenodoxin reductase (AdR) and cytochrome P450(scc) (CYP11A1) indicating the importance of this loop for the physiological function of this iron--sulfur protein.