(Received for publication, June 13, 1995)
From the e 10, D-13122 Berlin,
Germany and the
The amino acid in position 54 of adrenodoxin is strongly
conserved among ferredoxins, consisting of a threonine or serine. Its
role was studied by analyzing mutants T54S and T54A of bovine
adrenodoxin. Absorption, circular dichroism, fluorescence, and electron
paramagnetic resonance spectra of mutant T54S show that this
substitution has no influence on the formation and stability of the
ferredoxin. The redox potential of this mutant, however, was lowered by
55 mV as compared with native adrenodoxin, indicating a role for this
residue in redox potential modulation. Incorporation of the iron-sulfur
cluster was not impaired in the T54A mutant, although structural
features of the oxidized protein were considerably changed. The
decreased stability of the T54A mutant as compared with the wild type
and mutant T54S indicates that a hydrogen bond donor at this position
stabilizes the protein. Both mutants have been shown to be functionally
active. Replacement of threonine 54 by serine or alanine, however,
leads to rearrangements at the recognition sites for its redox
partners. This is reflected by decreased K and K
values of both mutants
for the cytochromes P450, whereas only T54A displayed a decreased K
value in cytochrome c reduction. Substrate conversion was accelerated (2.2- and 2.4-fold
for mutants T54A and T54S, respectively) in the CYP11B1-, but not in
the CYP11A1-dependent reaction.
Ferredoxins are ubiquitous iron-sulfur proteins present in
bacteria, plants, and animals. They take part in a broad variety of
electron transfer reactions. In plants and algae a ferredoxin of the
[2Fe-2S] type passes electrons from photosystem I to
NADP via a ferredoxin reductase in the process of
carbon assimilation(1) . The hydroxylase systems of vertebrates
and some bacteria utilize ferredoxin to transfer an electron from an
NAD(P)H-dependent reductase to various cytochromes P450 (2, 3, 4, 5) . The
[2Fe-2S] ferredoxins of bacteria, e.g. putidaredoxin, linredoxin, or terpredoxin, are components of the
hydroxylation systems for camphor, linalool, or
-terpineol,
respectively, the carbon sources of these
organisms(3, 4, 5) . In the adrenal cortex,
adrenodoxin is involved in steroid hormone biosynthesis. It passes
electrons to the mitochondrial cytochromes P450 CYP11A1, (
)CYP11B1, and CYP11B2, converting cholesterol to
pregnenolone, catalyzing the 11
-hydroxylation of 11-deoxycortisol
and 11-deoxycorticosterone, and producing aldosterone,
respectively(6, 7, 8, 9) .
The way in which the redox partners interact during electron transfer is still a matter of controversy. In the ``shuttle'' model(10, 11, 12, 13, 14, 15) , adrenodoxin sequentially forms binary complexes with the reductase and the cytochrome. A ternary complex of adrenodoxin reductase, adrenodoxin, and CYP11A1 is proposed in the second model(16, 17, 18, 19, 20) . Very recently, the necessity of two molecules of adrenodoxin for one-electron transfer has been suggested (21) which is supported by former results obtained with CYP11B1(22) .
Recognition and interaction of adrenodoxin with its redox partners is mainly based on electrostatic interactions(23, 24, 25) . However, tyrosine 82 of adrenodoxin was shown to participate in binding to CYP11A1 and CYP11B1, but not to adrenodoxin reductase(26) . Furthermore, transmission of conformational changes of the cluster to tyrosine 82 and the acidic residues of the binding region of adrenodoxin for its redox partners has been shown to be mediated by histidine 56(27) . Deletion of the C-terminal part of adrenodoxin up to amino acid 109 have been shown to influence binding affinity and electron transfer to CYP11A1 and CYP11B1, but not interaction with adrenodoxin reductase (28) .
The ability to accept and donate electrons is tightly connected to the redox potentials of iron-sulfur proteins. Generally, the features determining or modulating a certain redox potential are not well understood yet, but seem to be related at least in ferredoxins to the water accessibility of the iron-sulfur center, negative charges close to the cluster and on the protein surface, and hydrogen bonding pattern (extent, strength, and specific position)(29) . Some of these features should differ in the [2Fe-2S] proteins which cover a considerable redox potential range. The cysteine ligands to the cluster (cysteines 46, 52, 55, and 92 in adrenodoxin(30) ) are homologous in these proteins and therefore cannot account for their different properties. On the other hand, the single free Cys-95 in adrenodoxin is not able to replace Cys-92 when the latter residue was substituted by serine (30) as it was described for the [4Fe-4S] cluster of Azotobacter vinelandii ferredoxin I(31) . Replacement of any of the cysteine cluster ligands in adrenodoxin with serine lead to the formation of apoproteins(27, 30) . In contrast, the [2Fe-2S] ferredoxin of Clostridium pasteurianum(32) , Anabaena vegetative ferredoxin(33) , and the [2Fe-2S] center of the Escherichia coli fumarate reductase (34) are stable when the cysteine cluster ligands are replaced by serine and in the case of fumarate reductase also by aspartic acid(35) . So far, little is known about structures which stabilize the cluster and the conformation in ferredoxins(36) . Very recently, a method to facilitate calorimetric studies on folding and stability of adrenodoxin was developed(37) .
The different consequences of cluster ligand substitutions in adrenodoxin and other ferredoxins are of particular interest since the [2Fe-2S] center is assumed to adopt a similar tetrahedral structure in these proteins having homologous sequences around the ligand cysteines. The plant type ferredoxins display a higher degree of overall sequence similarity than the vertebrate type ferredoxins. As it is obvious from Fig. 1, the residue corresponding to position 54 of adrenodoxin is always occupied by either a threonine or a serine, residues which are known to be hydrogen bond donors within the protein or to water molecules that contribute to the protein stability.
Figure 1: Amino acid sequences of various vertebrate and plant type ferredoxins around three cysteine ligands to the [2Fe-2S] centers (positions 46, 52, and 55 of adrenodoxin). The conserved threonine/serine residue corresponding to position 54 in adrenodoxin is labeled. The cysteine cluster ligands are indicated by their position numbers in the ferredoxins. The sequences were taken from Refs. 5 (adrenodoxin, kidney ferredoxin, putidaredoxin, terpredoxin), 53 (placenta ferredoxin, Anabaena), 56 (S. oleracea, Equisetum, S. quaricauda, A. sacrum, N. muscorum), 62 (E. coli ferredoxin), and 63 (Pisum, S. platensis).
In order to understand the particular role of threonine 54 for the assembly of the iron-sulfur cluster and for determining its functional properties, we used mutants of adrenodoxin in which Thr-54 was replaced by serine (T54S) or alanine (T54A). These mutants were expressed in the cytoplasmic E. coli high-level expression system for adrenodoxin(30) , and their biochemical and biophysical characteristics were investigated in detail.
CD spectra were recorded on a
Jasco J720 spectropolarimeter. Samples contained 100 µM adrenodoxin in 10 mM potassium phosphate buffer (pH 7.4)
in a 1-cm cuvette for measurements in the 250-650 nm range and 20
µM adrenodoxin in 2 mM potassium phosphate buffer
(pH 7.4) in a 0.1-cm cuvette for measurements in the 184-260 nm
range. The spectrum of the respective potassium phosphate buffer was
recorded as a baseline (measurement conditions were: band width, 1 nm;
response, 2 s; step, 0.5 nm). Temperature-dependent measurements were
carried out at a heating rate of 50 °C/h from 20 to 80 °C with
a temperature increment of 0.2 degrees, monitoring the decrease of the
circular dichroism signal at 440 nm. Data were analyzed and T and
H
were determined
using a nonlinear regression program kindly provided by Dr. O. Ristau
(MDC, Berlin).
CYP11A1-dependent conversion of cholesterol to pregnenolone was performed as described(28) . Reaction mixtures consisted of 20 mM potassium phosphate buffer (pH 7.5), 0.3% Tween 20, 0.5 µM adrenodoxin reductase, 0.5 µM CYP11A1, 100 µM cholesterol, specified amounts of adrenodoxin, a NADPH regenerating system consisting of 600 µM glucose 6-phosphoric acid disodium salt, 4 units/ml glucose-6-phosphate dehydrogenase, and 60 µM NADPH. After the reaction, the steroids were converted into their corresponding 3-one-4-en forms by addition of 2 units/ml cholesterol oxidase, extracted, and analyzed by reverse-phase high performance liquid chromatography. Cholestenone and progesterone were used as external standards.
CYP11B1 assays contained 0.4 µM adrenodoxin reductase, 0.4 µM CYP11B1, 100 µM deoxycorticosterone, varying concentrations of adrenodoxin, the NADPH regenerating system, as described, in 50 mM potassium phosphate buffer (pH 7.5), 0.1 mM dithiothreitol, and 60 µM NADPH and were performed as described(28) . Dichloromethane, which acts to extract the steroids, was used to stop the reaction. The amount of corticosterone produced was determined by high performance liquid chromatography with corticosterone and 11-deoxycorticosterone as external standards.
Western blotting revealed specific bands (14 kDa each) for
adrenodoxin and the mutants (not shown). All proteins were expressed to
almost the same level (50-100 mg/liter of E. coli culture). The final purity index (A/A
) of the proteins was
always higher than 0.92.
Absorption spectra of oxidized [2Fe-2S] ferredoxins are
characterized by a number of absorption maxima in the visible and near
UV region. The typical peaks of adrenodoxin, caused by the
[2Fe-2S] cluster in its specific surrounding, occur at 455,
414, and 320 nm(6) . UV/vis spectra of wild type adrenodoxin
and mutant T54S were indistinguishable. In mutant T54A, however, a
bathochromic shift of the peak at 320 to 340 nm could be observed (Fig. 2). These apparent structural differences between the
oxidized forms of wild type adrenodoxin and mutant T54S on the one hand
and T54A mutant on the other hand were further analyzed by CD
spectroscopy since it sensitively reflects conformational changes in
optically active substances like a protein iron-sulfur cluster. In
fact, wild type adrenodoxin and mutant T54S again exhibit similar
spectra (Fig. 3, A-C), whereas mutant T54A
shows shifted CD signals and in part lower amplitudes of the peaks as
compared with wild type adrenodoxin. The signal changes in the
310-650 nm range clearly reflect a rearrangement in the immediate
vicinity of the cluster of this mutant (Fig. 3, A and B). Moreover, the T54A replacement seems to slightly affect
the general polypeptide backbone conformation (the local maximum at 195
nm shifts to 191 nm; Fig. 3C) and to display an
influence on the CD properties of the aromatic amino acids since the
molar circular dichroic absorption of this mutant decreases
from 1.4
10
(wild type) to -3.2
10
cm
mmol
at
297 nm (Fig. 3B). The fact that wild type adrenodoxin,
as well as the mutants, contain only a single tyrosine and four
phenylalanines, and lack tryptophan, suggests an involvement of the
tyrosine in the observed changes because phenylalanine residues are
known to contribute marginally to the spectral characteristics of a
protein. In order to confirm the latter observation, we recorded the
fluorescence spectra of the adrenodoxin mutants. While the tyrosine
fluorescence emission peak occurs at its characteristic position (305
nm) (46, 47) in both the wild type and the mutants,
the intensity of this peak is increased by 250% in mutant T54A as
compared with the wild type and mutant T54S (Fig. 4).
Figure 2: Absorption spectra of adrenodoxin and mutants T54S and T54A. The absorbance of the proteins was measured in the visible and UV range using a Shimadzu double-beam spectrophotometer UV2101PC. The concentrations of the proteins were 1.5 mM in 10 mM potassium phosphate buffer (pH 7.4).
Figure 3: CD spectra of adrenodoxin and the Thr-54 mutants. A, visible region; B, near UV region; C, far UV region. Samples consisted of 100 µM adrenodoxin in 10 mM potassium phosphate buffer (pH 7.4) for A and B, and 20 µM adrenodoxin in 2 mM potassium phosphate buffer (pH 7.4) buffer for C. wt, wild type.
Figure 4: Fluorescence emission spectra of Thr-54 mutants of adrenodoxin. The fluorescence emission of adrenodoxin, which contains a single tyrosine at position 82 and no tryptophan, was measured on a Shimadzu RF-5001 PC spectrofluorophotometer. Each sample contained 100 µM adrenodoxin in 10 mM potassium phosphate buffer (pH 7.4). The excitation wavelength was 275 nm. wt, wild type.
Figure 5:
Thermal denaturation curves of adrenodoxin
and the Thr-54 mutants. Single wavelength melting curves have
been recorded at 440 nm, the main peak of the iron-sulfur cluster in CD
spectroscopy, by slowly increasing the temperature from 20 to 80
°C. Thermal denaturation of adrenodoxin followed (A) in a
potassium phosphate buffer (pH 7.5) and (B) in a buffer system
containing 2-mercaptoethanol, NaS, and ascorbic acid in
glycine buffer (pH 8.5). wt, wild
type.
Substitution mutants of adrenodoxin in which threonine 54 was replaced by serine or alanine were analyzed in order to study the role of this amino acid residue located between two cysteine ligands to the [2Fe-2S] cluster at a position which is strongly conserved (threonine or serine) among the ferredoxins of bacteria, plants, and animals. Attention has especially been paid to structural properties and stability of the mutants by analyzing their spectral features, to their redox potentials, and to their electron acceptor and donor functions.
The specific environment of a certain reduced
[2Fe-2S] cluster is sensitively reflected by EPR spectroscopy
as confirmed by the different EPR spectra of plant and vertebrate type
[2Fe-2S] proteins, although their general structure is
assumed to be similar. The EPR signals of the adrenodoxin mutants
resemble those of the wild type and therefore indicate that the
conformation of the reduced iron-sulfur center in adrenodoxin is not
significantly affected by the substitutions at position 54 (data not
shown). The structural properties of the proteins in oxidized form,
however, point to the necessity of a hydroxyl group at position 54 for
maintaining the native conformation of the oxidized iron-sulfur center.
While mutant T54S and wild type adrenodoxin exhibit similar
characteristics in optical, CD, and fluorescence spectroscopy (Fig. 2Fig. 3Fig. 4), substitution of the
hydrophobic alanine at position 54 (mutant T54A) causes a rearrangement
in the environment of the oxidized cluster ( Fig. 2and Fig. 3). This T54A mutation also affects the remote tyrosine
residue near the negatively charged protein surface of adrenodoxin
which is involved in binding of adrenodoxin reductase and cytochrome
P450 (Fig. 4), and slightly influences the polypeptide backbone
conformation (Fig. 3C). Alanine is not able to form a
hydrogen bond as is the native threonine, and thus destabilizes the a priori highly flexible cluster area (52) of a
ferredoxin by making it even more flexible, whereas a hydrogen bonding
system would not significantly be disturbed by a TS replacement.
The diminished volume of serine as compared with threonine in mutant
T54S does not lead to any significant spectral changes.
The missing
hydrogen bond and the rearrangement of the iron-sulfur cluster
surrounding in mutant T54A results in a decrease of the thermal
stability of this protein. Temperature-dependent CD measurements reveal
lower T values (5.5 and 2.6 degrees; Table 1) for mutant T54A in both buffer systems used. Also, the
H
value of this mutant for the reversible
denaturation process is lowered by 51 kJ/mol as compared with that of
the wild type (Table 1). The stability of mutant T54A upon
storage is also diminished as compared with the wild type, as indicated
by a decrease in the absorbance ratio A
/A
from >0.9 to
0.8.
The three-dimensional structures of [2Fe-2S]
ferredoxins carrying a threonine (53, 54) or a serine (55) at the position equivalent to threonine 54 in adrenodoxin
allow speculation about the role of this residue in adrenodoxin. In
putidaredoxin(53) , T47-OH (being homologous to threonine 54 in
adrenodoxin) is excluded from water molecules and located close (3
Å) to the cluster ligand C45-S
, suggesting a contact between
these two amino acid residues. A similar hydrogen bond between T48-OH
and C46-S
(3.4 Å) was proposed for the [2Fe-2S]
ferredoxin from the cyanobacterium Anabaena(52, 54) . Other authors show that
T48-OH is exposed to a water molecule (3.15 Å)(56) , as
was discussed for Equisetum arvense ferredoxin I, in which
serine 45 occupies the position corresponding to threonine 54 in
adrenodoxin(55, 57) . Another type of involvement, a
NH
S bond between T48-NH and C46-S
, was proposed
for the ferredoxin of the blue-green alga Spirulina
platensis(58) . All these contacts seem to confer a
stabilizing effect on the iron-sulfur center. The general structures of
these proteins are similar in this region, with putidaredoxin showing
the greatest similarities to adrenodoxin regarding its amino acid
sequence (33% sequence identity), spectroscopic properties, and the
interprotein electron transfer function in the respective hydroxylase
system. A model of adrenodoxin based on the NMR model of putidaredoxin
proposes a distance of only 2.96 Å between T54-OH and the cluster
ligand C52-S
(Fig. 6).
Figure 6:
Three-dimensional model of the
[2Fe-2S] cluster region of adrenodoxin based on NMR data of
putidaredoxin. The cluster ligand C52-S and T54-O
are
depicted as sticks and balls. Besides being located
close to C52-S
(2.96 Å), threonine 54 seems to occupy a
position which connects the environment of the cluster with the surface
of the protein.
While the replacement of threonine 54 by serine does not affect the conformation of the cluster in its environment, the redox potential of mutant T54S is markedly lowered (Table 2) in comparison to wild type adrenodoxin. Interestingly, the redox potentials of the [2Fe-2S] proteins of higher plants, which contain a serine at the position corresponding to threonine 54 of adrenodoxin (the only known exception is Equisetum ferredoxin II, see Fig. 1), are usually 50 mV lower than those of most ferredoxins from blue-green algae, in which this position is occupied by a threonine (except for Anabaena heterocyst ferredoxin, see Fig. 1)(59) , although both groups belong to the class of plant type ferredoxins(33, 59) . Substitution of T54S in adrenodoxin caused a similar redox potential shift (-55 mV; Table 2) leading to the conclusion that the residue naturally occurring at this position (threonine or serine) is directly involved in redox potential tuning. Determination of the redox potential of mutant T54A revealed a value 11 mV lower than that of mutant T54S (Table 2). Alanine as a non-aromatic apolar residue has a low surface probability resulting from its hydrophobicity. It is effective in excluding the solvent close to the iron-sulfur center, thus leading to a low dielectric constant and a decrease in the redox potential of the protein(60) . A generally higher redox potential as displayed by the vertebrate-type ferredoxins in comparison to plant-type ferredoxins permits a higher concentration of reduced [2Fe-2S] centers during turnover(61) , which might be essential to prevent competition between oxidized and reduced ferredoxins for binding to its electron acceptor as it was described for adrenodoxin and CYP11A1(14, 28) .
The redox potential is closely related to the electron acceptor and donor functions of adrenodoxin, which are retained in the mutants. Upon complex formation with CYP11A1, both mutants were able to shift their redox potentials to values close to that of the native protein (-291 mV(12) ; Table 2), indicating that the redox potential of the free adrenodoxin does not determine the electron transfer rate in the CYP11A1-dependent reaction.
The capability of adrenodoxin to transfer electrons was not impaired by substitution of threonine 54. The electron acceptor function of oxidized adrenodoxin mutants was tested in a cytochrome c reduction assay. There are only marginal changes in the kinetic parameters indicating that replacement of threonine 54 by serine or alanine does not considerably affect interaction with adrenodoxin reductase.
The affinity of adrenodoxin to CYP11A1 (Table 5) and the hydroxylating activities of CYP11A1 and CYP11B1 (Table 3), however, were particularly affected by the T54S mutation, while mutant T54A showed less pronounced effects. This points to either different binding sites for adrenodoxin reductase and the cytochromes or the possibility that interaction with the cytochromes more sensitively reflects minimal changes of the adrenodoxin molecule. It is unlikely that threonine 54 interacts directly with cytochrome P450, as can be suggested from the three-dimensional models of adrenodoxin. However, threonine 54 seems to occupy a position which connects the environment of the cluster with the surface of the protein (Fig. 6). Furthermore, the efficiency of the cholesterol side chain cleavage with the threonine 54 mutants is nearly unchanged, whereas the conversion rate of 11-deoxycorticosterone to corticosterone using these mutants is accelerated more than 2-fold as compared with the wild type (Table 3), pointing to an enhanced velocity of the second electron transfer to CYP11B1. The first electron transfer to CYP11B1, but not to CYP11A1, was also slightly accelerated using mutants T54S and T54A (Table 4). A similar discrimination between CYP11A1 and CYP11B1 was already described(28) . In contrast, the ability to accept and donate electrons was not significantly affected by replacement of threonine 48 (the homologous residue to threonine 54 in adrenodoxin) by either serine or alanine in Anabaena vegetative ferredoxin(52) , indicating that the electron transfer rate is differentially regulated in plant and vertebrate type ferredoxins.
In conclusion, the alcoholic residue at the position corresponding to residue 54 in adrenodoxin plays an important role in determining the redox potential of [2Fe-2S] proteins. Introduction of the more hydrophobic alanine at this position in adrenodoxin markedly decreases the stability of the holoprotein, indicating participation of the hydroxyl group of residue 54 in stabilizing the cluster by hydrogen bonds. Additionally, substitution of threonine 54 by alanine leads to changes in the spectral features of the oxidized protein, but not in the reduced state, indicating a remarkable rearrangement of the cluster surrounding upon reduction of the protein. Both mutants retained the functional capabilities of adrenodoxin. The observed variations in the kinetic parameters reflect the fact that substitution of threonine 54 results in a slight rearrangement at the surface of adrenodoxin and provide further evidence for discrimination not only between the reductase and the cytochromes, but also between CYP11A1 and CYP11B1. The mechanisms which control the electron transfer rates to the different cytochromes P450 and in other ferredoxin-dependent redox systems are subject to further studies.