From Fachrichtung 8.8 Biochemie, and Fachrichtung 8.7 Pharmakognosie und Analytische Phytochemie, Universität des
Saarlandes, D-66041 Saarbrücken, Germany
Received for publication, August 21, 2000
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ABSTRACT |
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The amino acid in position 49 in bovine
adrenodoxin is conserved among vertebrate [2Fe-2S] ferredoxins as
hydroxyl function. A corresponding residue is missing in the
cluster-coordinating loop of plant-type [2Fe-2S] ferredoxins. To
probe the function of Thr-49 in a vertebrate ferredoxin, replacement
mutants T49A, T49S, T49L, and T49Y, and a deletion mutant, T49 [2Fe-2S] ferredoxins are found in all organisms from archaea and
bacteria to higher plants and animals and function as mediators of
electron transfer in a range of multicomponent redox systems (1). The
redox active prosthetic group in this class of ferredoxins is
characterized by an iron-sulfur cluster, consisting of two non-heme
iron ions ligated to thiolate side chains of four cysteines of the
polypeptide, bridged by two inorganic sulfide ions.
[2Fe-2S] ferredoxins are classified by structure and function into
plant-type ferredoxins and vertebrate-type ferredoxins. Vertebrate-type
[2Fe-2S] ferredoxins, present in oxygenase systems of bacteria and
vertebrates, transfer electrons from a NAD(P)H-dependent ferredoxin reductase to different cytochrome P450 enzymes (2). In
vertebrates, ferredoxins of the [2Fe-2S] protein family are present
in adrenal cortex, placenta, liver, kidney, and brain (3), where they
participate in cytochrome P450-catalyzed hydroxylation reactions to
produce steroid hormones, vitamin D metabolites, and bile acids.
Bovine Adx1 as a member of
vertebrate [2Fe-2S] ferredoxins, functions as electron mediator from
the isoalloxazin system of adrenodoxin reductase (AdR) to the heme iron
of two cytochromes P450, CYP11A1 and CYP11B1, localized in the inner
mitochondrial membrane of the adrenal cortex (4). CYP11A1 converts
cholesterol to pregnenolone, and CYP11B1 catalyzes the
11 The crystal structures of a truncated bovine Adx (5) and of a
full-length Adx (6) determined at 1.85- and 2.5-Å resolution, respectively, display a compact ( The ability to accept and donate electrons during interaction with
redox partners is tightly connected to the redox potentials of
iron-sulfur proteins. The redox potential differs widely in [2Fe-2S]
proteins and is correlated to the ferredoxin type. In general,
plant-type ferredoxins display lower redox potentials (between Expression and Mutagenesis--
Adx proteins were produced using
the T7-expression system consisting of E. coli strain
BL21[DE3]pLysE and vector pET3d (19). Mutations in the Adx cDNA
were introduced by polymerase chain reaction according to Landt
et al. (20). Oligonucleotides containing the appropriate
restriction sites and mutations were synthesized by BioTez GmbH.
Protein Purification--
Recombinant Adx and AdR were purified
as described (21, 22). Protein concentration was calculated using
Spectroscopic Methods--
Absorption spectra in the UV-visible
region were recorded at room temperature on a Shimadzu double-beam
spectrophotometer UV2101PC.
Samples for NMR spectroscopy (5 mg each) of Adx and T49
CD spectra were recorded as described previously (26) on a Jasco 715 spectropolarimeter. Temperature-dependent measurements were
carried out at a heating rate of 50 °C/h from 20 to 65 °C with a
temperature increment of 0.2 °C, monitoring the decrease of the
circular dichroism signal at 440 nm. Tm and
Hm were calculated from CD scans with a
nonlinear regression program using the two-state model (27).
Redox Potential Measurements--
Redox potentials of Adx and
mutants were determined using the dye photoreduction method (28) with
safranin T as indicator and mediator as described.
Optical Difference Spectroscopy--
Binding of Adx and mutants
to CYP11A1 was followed spectrophotometrically as a high spin shift of
the P450 heme iron from 417 nm to 393 nm, caused by Adx-induced
cholesterol binding (29). Titrations were performed in tandem cuvettes
with 2 µM CYP11A1, 25 µM cholesterol,
0.03% Tween 20 in a buffer containing 50 mM potassium
phosphate, pH 7.4, 0.1 M potassium chloride, 0.1 mM EDTA, 0.1 mM dithioerythritol, 0.05%
sodium cholate at room temperature. Calculation of free Adx was done as
described by Coghlan and Vickery (30).
Stopped-flow Experiments--
Measurements of the first electron
transfer reaction from Adx to cytochrome CYP11A1 were carried out using
a single channel stopped-flow ASVD spectrophotometer SX-17MV (Applied
Photophysics). The reduction of cytochrome CYP11A1 in a buffer
containing 50 mM potassium phosphate pH 7.4, 0.1 M potassium chloride, 0.1 mM EDTA, 0.1 mM dithioerythritol, 0.05% sodium cholate was
followed at 15 °C by monitoring the absorption at 450 nm,
representing the formation of the ferrous-carbon monoxide complex
(31).
Enzyme Activity Assays--
The interaction of Adx and AdR was
assayed following the reduction of cytochrome c in a buffer
containing 50 mM potassium phosphate (pH 7.4) and 10 mM potassium chloride. The reaction was initiated by
addition of NADPH. The absorption change at 550 nm was monitored, and
the activity determined using an extinction coefficient of 20 (mM cm) Calculation of Electron Coupling--
The tunneling pathway
model of Beratan (33) was used to calculate the electron transfer from
the [2Fe-2S] cluster to the ferredoxin surface. The program Pathway
(version 0.97) classifies the pathways into covalent, hydrogen-bonded,
and through-space (van der Waals contact) segments depending on the
kind of interaction between the neighbored atoms, and finds optimal
pathways with maximal coupling between electron donor and acceptor.
Expression of Mutants--
To confirm that
oligonucleotide-directed mutagenesis was limited to the predicted
sites, Adx cDNA inserts in the expression vector pET3d were
sequenced using the cycle sequencing method. All Adx proteins were
expressed as holoproteins in the cytoplasm of E. coli strain
BL21[DE3]pLysE. SDS-PAGE and Western blotting revealed specific bands
at 14 kDa for all proteins. The expression yields of mutants T49A,
T49S, T49L, and T49Y were each in the range of wild type adrenodoxin
(50-100 mg/liter of E. coli culture). Yields of mutant
T49 CD Spectroscopy--
To measure the effect of mutations on the
structure of the iron-sulfur cluster region CD spectroscopy was
applied, a method that sensitively detects conformational changes in
optically active substances like iron-sulfur cluster containing
proteins. No significant changes were observed either in the near UV or
visible region for the substitution mutants (Fig.
2). In contrast, deletion mutant T49 NMR Spectroscopy--
To study the mutational effect of the
deletion of Thr-49 on the aromatic region of T49 Thermal Denaturation--
To analyze the effect of mutations on
the protein stability, thermal unfolding of Adx was performed. A buffer
system that prevents the destruction of the [2Fe-2S] cluster during
heating (35) allows unfolded proteins to regain the original absorption spectrum after renaturation (36). Wild-type Adx and all substitution mutants renatured after a 12-h incubation at 4 °C in the
sulfide-containing buffer. In contrast, mutant T49
The CD signal of melting curves of all analyzed proteins recorded at
440 nm decreased in a sigmoidal manner (Fig.
4), allowing a data fit according to a
two state model. The thermal transition temperatures
Tm of mutants T49A, T49S, and T49L are nearly
unchanged. Tm values of mutants T49Y and T49
In order to analyze the stability of mutant T49 Redox Potential--
The redox potential of an electron transport
protein indicates its ability to accept and donate electrons. The
midpoint redox potentials for wild type Adx and mutant ferredoxins were
analyzed by spectrophotometric redox measurements using safranin T as
indicator and mediator. Substitutions of the amino acid in position 49 did not affect the redox properties of the proteins (Table
II). The measured redox potential of wild
type Adx and the replacement mutants show an almost identical value of
Affinity to the Redox Partner AdR--
In order to determine the
extent to which the mutations affect the interaction with AdR, an assay
involving cytochrome c as an artificial electron acceptor
was used. Under the conditions employed, cytochrome c is in
large excess, and the derived Km values are
essentially equivalent to dissociation constants
(Kd) of the AdR-Adx complex (39). Table
III shows that the affinity of the
mutants T49A, T49S, and T49L to AdR is in the same range as the wild
type. Replacement of Thr-49 with tyrosine resulted in a 3-fold increase
in complex formation, whereas the deletion of Thr-49 caused a 120-fold
increase of the Km value.
Affinity to the Redox Partner CYP11A1--
Binding of the oxidized
form of adrenodoxin to its electron acceptor CYP11A1 is comparable to
that of the reduced form (40), thus allowing to analyze the affinities
of wild type and mutant adrenodoxin to CYP11A1 by optical difference
spectroscopy. Compared with wild type Adx, mutants T49S, T49L, and T49Y
exhibit Kd values in the same range, mutant T49A
shows a nearly 8-fold increased Kd value, and the
Kd value of mutant T49 Reconstitution of Enzymatic CYP11A1 Activity--
The effect of
mutations in position 49 on the enzymatic activity of the native
electron acceptor CYP11A1 was investigated by HPLC analysis of the
substrate conversion of cholesterol into pregnenolone (Table III). The
values for maximal substrate conversion, Vmax,
are quite similar for the wild type and the mutants T49A, T49S, and
T49Y. The Vmax value for mutant T49L is
decreased by 30%. A substrate conversion with the deletion mutant
T49 Reduction of CYP11A1-Substrate Complex by Adx--
To investigate
the effect of mutations at position 49 on the electron transfer rate to
CYP11A1, stopped-flow experiments in oxygen-free atmosphere were
performed. In the experiments adrenodoxin, preincubated with AdR and
NADPH in the first test tube, was rapidly mixed with oxidized CYP11A1
present in the second test tube. Under these conditions the rate
constants of electron transfer from the reduced Adx to the oxidized
CYP11A1 are fully Adx-dependent since the flavin to
iron-sulfur electron transfer is not rate-limiting. The apparent rate
constant, kapp, measured for wild type Adx is 0.098 s Calculation of the Electron Coupling Map of Adx--
A global
coupling map of the Adx molecule was calculated with the program
Pathway (version 0.97) using Fe-1 of the [2Fe-2S] cluster as donor
and all other atoms as acceptors. The coupling map was displayed on the
surface of the three-dimensional structure of the truncated mutant of
adrenodoxin, consisting of amino acids 4-108, with the program
InsightII (Fig. 6). A small region with high coupling values can be identified on the surface of the molecule. All amino acids with high coupling are located in the loop covering the
iron-sulfur center comprising the residues between position 47 and 54. The highest coupling values were calculated with the program Harlem for
the residues Thr-54 (6.4 × 103), Thr-49 (5.3 × 103), Leu-50 (4.0 × 103), and Glu-47
(3.7 × 103), indicating a high probability that these
amino acids are involved in electron transfer from the redox active
iron-sulfur cluster to the Adx surface.
The importance of the threonine residue in position 49 in bovine
Adx for its function as electron mediator has been investigated using
site-directed mutagenesis and detailed analysis of the obtained mutants. Special attention has been attributed to structural and stability properties, to the redox potential, to the electron transfer
function, and to the influence on binding affinities to the redox partners.
In the mature Adx, Thr-49 is located in a 5-residue-containing loop
between the [2Fe-2S] cluster coordinating cysteines Cys-46 and
Cys-52. This amino acid is conserved in [2Fe-2S] ferredoxins of the
vertebrate-type as threonine or serine. In plant-type ferredoxins a
corresponding residue is missing, and the iron-sulfur cluster loop
contains only 4 residues.
To determine the influence of amino acid substitutions or a deletion in
position 49 on the structural properties of the protein, CD spectra
have been recorded. As concluded from unchanged spectra, the
introduction of alanine, serine, leucine, or tyrosine instead of
threonine did not lead to changes in the environment of the iron-sulfur
cluster. This observation is in agreement with computer modeling
studies, which indicated that the introduction of the respective amino
acids does not cause any steric hindrance in its surrounding (data not
shown). Differences in the spectroscopic properties of wild type Adx
and mutant T49 For further analysis of the extent of the apparent structural changes
of mutant T49 In the tertiary structure of Adx, the iron-sulfur redox center is
located close to the protein surface, where Thr-49 is positioned at the
boundary separating the redox center from the aqueous phase (Fig.
7). Several potential hydrogen bonds
around the iron sulfur cluster have been identified (5). In the
immediate vicinity of Thr-49, the backbone amide groups of Glu-47 (E47N
to S1), Gly-48 (G48N to C46S, were
generated and expressed in Escherichia coli. CD spectra of
purified proteins indicate changes of the [2Fe-2S] center geometry
only for mutant T49
, whereas NMR studies reveal no transduction of
structural changes to the interaction domain. The redox potential of
T49
(
370 mV) is lowered by ~100 mV compared with wild type
adrenodoxin and reaches the potential range of plant-type ferredoxins
(
305 to
455 mV). Substitution mutants show moderate changes in the binding affinity to the redox partners. In contrast, the binding affinity of T49
to adrenodoxin reductase and cytochrome P-450 11A1
(CYP11A1) is dramatically reduced. These results led to the conclusion
that Thr-49 modulates the redox potential in adrenodoxin and that the
cluster-binding loop around Thr-49 represents a new interaction region
with the redox partners adrenodoxin reductase and CYP11A1. In addition,
variations of the apparent rate constants of all mutants for CYP11A1
reduction indicate the participation of residue 49 in the electron
transfer pathway between adrenodoxin and CYP11A1.
INTRODUCTION
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DISCUSSION
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-hydroxylation of 11-deoxycorticosterone and 11-deoxycortisol and
the production of aldosterone.
+
) fold typical for [2Fe-2S] ferredoxins. The polypeptide chain is organized into a large core domain, containing the iron-sulfur cluster, and a smaller interaction domain. This small 35-amino acid-comprising domain includes the acidic
region between residues 72 and 79, which was shown to be responsible
for the recognition of the redox partners AdR and cytochrome CYP11A1
(7). The identification of corresponding positively charged residues on
the interaction partners AdR and CYP11A1 (8, 9) confirmed a recognition
model mainly based on electrostatic interactions.
305
and
455 mV) (10) than vertebrate-type ferredoxins (
235 to
273
mV). The protein sequence alignment of the region around the [2Fe-2S]
cluster ligands (Fig. 1) between plant-type ferredoxins and
vertebrate-type ferredoxins displays a difference between the two
ferredoxin types in the length of the metal-binding loop proximate to
the reducible iron atom. The loop in vertebrate-type ferredoxins
contains five residues, and the conserved alcoholic amino acid in the
middle of the loop consists of a threonine or serine (Fig.
1). In plant-type ferredoxins, the loop
is one amino acid shorter and a corresponding conserved residue is
missing. In Adx, a threonine is positioned in the middle of the loop
between the cysteine cluster ligands Cys-46 and Cys-52. In order to
understand the particular role of the amino acid Thr-49 for the
functional properties of the [2Fe-2S] cluster, a series of mutants of
Adx was generated in which Thr-49 was replaced by alanine (T49A), serine (T49S), leucine (T49L), and tyrosine (T49Y) or Thr-49 was deleted (T49
). Recombinant proteins were purified from E. coli and characterized in detail by biochemical and biophysical
methods.
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Fig. 1.
Amino acid sequence alignment around three
cysteine ligands to the iron-sulfur cluster loop. The cysteine
ligands of the iron-sulfur center (positions 46, 52, and 55 in Adx) are
marked in gray. The position corresponding to Thr-49 in Adx
is indicated in bold type. The ferredoxin
sequences were from cattle (Adx) (11), Pseudomonas
putida (Pdx) (12), Pseudomonas sp.
(Terpredoxin) (13), E. coli (Fdx
Eco) (14), Equisetum arvense (Fdx
Ear) (15), Aphanothece sacrum (Fdx
Asa) (16), Anabaena sp. (Fdx
Ana) (17), and Haloarcula marismortui
(Fdx Hma) (18).
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DISCUSSION
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414 = 9.8 (mM cm)
1
for Adx (23) and
450 = 11.3 (mM
cm)
1 for AdR (24). Isolation of CYP11A1 and
CYP11B1 from bovine adrenal glands was performed according to Akhrem
et al. (25) with slight modifications.
in 10 mM potassium phosphate buffer were dried at room
temperature under vacuum and dissolved in 0.5 ml of D2O.
The pH was adjusted to 8.2 with a 50 mM potassium phosphate
buffer. Both samples were left at 4 °C for approximately 72 h
to exchange the amide protons for deuterium. 1H NMR spectra
were recorded on a Bruker DRX 500 NMR spectrometer at 299 K. The
residual water signal was suppressed with low power irradiation.
1 for cytochrome
c. The cholesterol side chain cleavage activity was assayed
in a reconstituted system catalyzing the conversion of cholesterol to
pregnenolone according to the procedure of Sugano et al.
(32). Assays were performed at 30 °C in 50 mM potassium phosphate, pH 7.4, 0.1% Tween 20 and contained a NADPH regenerating system. The samples were analyzed by reversed phase HPLC with an
isocratic solvent system of acetonitrile/isopropanol (15:1).
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were reduced to one third of the wild type level. The final
purity index (A414/A276)
of the proteins was always higher than 0.9.
showed in the indicated regions shifted CD signals and in part lower
amplitudes of the peaks compared with the wild type. Deletion of Thr-49
shifted the local maxima at 440 and 342 nm to 428 and 348 nm,
respectively. In addition, a new shoulder appeared at 455 nm in the
T49
spectrum. The signal changes in the range of 310-650 nm clearly
reflect a rearrangement in the proximity of the iron-sulfur cluster of
this mutant.
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Fig. 2.
CD spectra of Adx and Thr-49 mutants.
Spectra in the visible and near UV region are shown for wild type Adx
(solid line), T49 (long
dashed line), T49A (short dashed
line), T49S (dash-dot line), T49L
(dash-dot-dot line), and T49Y (dotted
line). Samples consisted of 40 µM Adx in 10 mM potassium phosphate buffer (pH 7.4).
, 1H NMR
spectra of T49
and Adx were recorded under the same conditions and
compared with each other (Fig. 3). Both
spectra show the same chemical shifts for the various aromatic residues
(assignment according to Beckert et al. (Ref. 34)),
indicating that the three-dimensional global structure of the protein
was maintained after deletion of the amino acid Thr-49.
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Fig. 3.
One-dimensional 1H NMR spectra of
Adx and mutant T49 . Spectra of Adx
(A) and mutant T49
(B) were recorded at pH 8.2 after deuterium exchange.
denatured irreversibly.
are 2.5 °C and 13.3 °C lower compared with the wild type (Table
I). The denaturation enthalpy
dHm seems to be correlated with
the size of the substituting amino acid (Fig.
5). The values for the
dHm differences in kJ/mol decrease in the row T49A (+24), T49S (+2), wild type (0), T49L (
9),
and T49Y (
14).
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Fig. 4.
Thermal denaturation curves of Adx and Thr-49
mutants. Single wavelength melting curves have been recorded by CD
spectroscopy at 440 nm. Thermal denaturation of wild type Adx
(solid line), T49 (long
dashed line), T49A (short dashed
line), T49S (dash-dot line), T49L
(dash-dot-dot line), and T49Y (dotted
line) were followed in a glycine buffer system (pH 8.5) containing
2-mercaptoethanol, Na2S, and ascorbic acid.
Thermodynamic parameters of unfolding for wild type Adx and Thr-49
mutants
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Fig. 5.
Correlation of the denaturation enthalpy of
Adx mutants with the size of the amino acid in position 49. The
calculated thermal denaturation enthalpy
dH(Td) for wild type
(wt) Adx and mutants in position 49 is shown. The
inset presents the dependence of the
(
dH(Td)) values on
the side chain volume of the amino acid present in position 49 (correlation coefficient 0.7). Side chain volumes of amino acids were
taken from Zamyatin (37).
in the respective
buffer system of different functional assays, absorption spectra were
recorded before and after the indicated incubation time. The spectral
properties of the mutant remained nearly unchanged in all buffer
systems used (spectra not shown).
276 mV, which resembles the potential for the native Adx isolated
from bovine adrenal glands (38). The value for the deletion mutant
T49
(
370 mV) was about 100 mV lower than that measured for the
wild type protein, indicating a significant role of this position in
modulating the redox potential.
Redox potentials of Adx and Thr-49 mutants
Kinetic constants of adrenodoxin and Thr-49 mutants
was not detectable (Table
III).
was not detectable. The Km values for the
substrate conversion are similar for the wild type and mutant T49S and
slightly increased for mutants T49A, T49L, and T49Y, whereas no
Km for the reaction with the mutant T49
can be calculated.
1 (Table
IV). The value for mutant T49S is
slightly increased (0.140 s
1), whereas the
value for mutant T49A is slightly decreased (0.063 s
1). The rate constants for mutants T49L and
T49
are 3- and 6-fold reduced, respectively. Substitution of the
threonine in position 49 by a tyrosine resulted in a
kapp value increased by a factor of 3.
Reduction rate of CYP11A1
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Fig. 6.
Electronic coupling between the iron-sulfur
cluster and the surface atoms in the Adx molecule. A global
electron coupling map was calculated with the program Pathway (version
0.97). The Fe-1 of the [2Fe-2S] cluster was set as electron
donor, and all other atoms and bonds functioned as electron acceptors.
The calculated data were transferred to the protein structure of Adx
and visualized with the program InsightII (MSI Inc.). Red
indicates a high coupling rate; blue represents a low
coupling rate. The amino acid Thr-49 is indicated on the surface in
white.
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were discernible in the CD spectra of the proteins.
Significant changes in both the near-UV and visible region sensitively
reflect conformational changes in the surrounding of the optically
active iron-sulfur cluster of mutant T49
. The CD spectrum of this
mutant shows shifted CD signals, in part lower amplitudes of the peaks
compared with the wild type, and the appearance of a new shoulder at
455 nm in the region of the largest CD maximum. Thus, shortening of the cluster loop by only one amino acid causes rearrangements in the active
center, whereas the CD signals of substitution mutants seem not to be
sensitive to a larger or smaller side chain volume of the amino acid in
position 49.
, 1H NMR spectroscopy was applied. The
1H assignments of residues His-10, His-56, His-62, and
Tyr-82 have been confirmed as obtained previously (34) and display the
same chemical shift in wild type Adx and in the mutant T49
. Residue Tyr-82 is part of the interaction domain, and residue His-56 forming hydrogen bonds to Tyr-82 and Ser-88 connects the core domain with the
interaction domain and transmits conformational changes upon reduction
of Adx. Since NMR signals of both residues indicate the same chemical
environment in the mutant and in the wild type, a transduction of
conformational changes in the cluster region of mutant T49
to the
interaction domain can be excluded. The deletion, therefore, has no
obvious effect influencing the structure around the residues on the
acidic domain described as critical for the interactions studied.
), and Ala-51 (A51N to C46S
) may form
a hydrogen bond to the sulfur atom S1 or to the sulfur atom of Cys-46
of the iron-sulfur cluster. The importance of these hydrogen bonds was
directly studied by thermal unfolding of the proteins in a CD
spectropolarimeter. The nearly unchanged conformational stability of
the iron sulfur cluster in substitution mutants compared with wild type
Adx is in agreement with the suggestion that the hydrogen bond network
in the neighborhood of position 49 was not affected by the mutations.
In contrast, the reduced Tm value for the
deletion mutant indicates that the stability of the iron-sulfur cluster vicinity is dramatically affected by lost or by weaker hydrogen bonds.
This finding is supported by the irreversibility of the unfolding of
mutant T49
, which is in contrast to reversible folding properties of
the other Adx mutant proteins.
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Fig. 7.
Three-dimensional structure of the
iron-sulfur loop region in Adx. Key residues discussed in the text
are labeled.
The deletion of the threonine in position 49 and the accompanying
distortion of the hydrogen bond network results in a dramatic redox
potential drop from 276 mV (wild type) to
370 mV (T49
). In
contrast, all substitution mutants display an unchanged midpoint redox
potential. The results of redox potential determinations suggest that
the amino acid in position 49 stabilizes the hydrogen bond network and
tunes the microenvironment of the iron-sulfur cluster and therefore the
redox potential in bovine Adx. An effect of hydrogen-bonding
interactions between [2Fe-2S] cluster ligand atoms and side chain or
main chain donor atoms on the oxidation-reduction potentials of Adx
(41) and Anabaena ferredoxin (42) has been observed
previously. The redox potentials of bovine Adx mutants T54A and T54S
are reduced by 55 and 34 mV, respectively. In this case, a hydrogen
bond from the side chain hydroxyl group of Thr-54 to the sulfur atom of
Cys-52 is affected in the mutant proteins and results in structural
changes in the vicinity of the iron-sulfur cluster. In
Anabaena ferredoxin mutant A45S, the presence of an additional hydrogen bond in the metal-binding loop from the side chain
hydroxyl group of Ser-45 to the sulfur atom of Cys-41 seems to be
responsible for the higher midpoint potential of mutant ferredoxin
(
382 mV) relative to the wild type (
406 mV). The redox potential
for the Adx deletion mutant T49
(
370 mV) reaches the potential
range of plant-type ferredoxins (
305 to
455 mV), which is in
general lower as in vertebrate-type ferredoxins. Since the deletion in
mutant T49
results in a 4-residue-containing [2Fe-2S] cluster loop
as present in plant-type ferredoxins, similar structures may be formed,
which are a prerequisite to determine the redox potential in this
range. Further crystallographic studies solving the detailed structure
of the [2Fe-2S] center vicinity of the mutant T49
will present
more insight into the general field of redox potential determination in ferredoxins.
The affinity of Adx to the redox partners CYP11A1 and AdR is
particularly affected by mutations in position 49. The most pronounced effects can be observed for the deletion mutant T49, which shows a
120-fold decrease in binding affinity to AdR and a drop to zero in the
binding affinity to CYP11A1. Smaller effects were measured after
replacement of Thr-49 with tyrosine or alanine. Mutant T49Y shows a
3-fold increase in complex formation with AdR, and mutant T49A shows an
8-fold decrease in complex formation with CYP11A1. Measurement of the
binding constants of the other mutants to AdR and CYP11A1 revealed only
slight changes or similar results compared with the wild type. Since
binding constants for the substitution mutants are only changed for the
largest and smallest introduced residues (T49A, T49Y), the surface
contact via Thr-49 seems to be optimized for interaction with different
redox partners. Dramatically changed binding properties of the deletion
mutant, caused by structural rearrangements in the cluster region,
suggest a crucial role of Thr-49 for direct interaction with binding
partners or for the formation of an interacting surface represented by
the [2Fe-2S] cluster loop. Corresponding to Thr-49 in Adx, in the
bacterial ferredoxin putidaredoxin (Pdx) residue Ser-44 was substituted in a mutagenesis study (43). The authors suggested that substitution of
Ser-44 with the smaller amino acid, glycine, increases solvent accessibility to the cluster and simultaneously induces distortion around the ferrous iron as indicated by shifting the though in the
g
electron spin resonance signal to a higher
magnetic field. This mutation causes the same spectral changes of Pdx
as the formation of the ternary complex Pdx-CYP101-CO. In the complex the change in the CYP101 active site upon CO binding is transmitted to
Pdx within the complex and produces a conformational change of the
iron-sulfur active center. Ser-44 in Pdx, homologous to Thr-49 in Adx,
is part of a surface loop around the [2Fe-2S] center including the
residues Asp-34, Asp-38, and Ser-42 involved in redox partner binding
(44). Both proteins, the vertebrate ferredoxin Adx and the bacterial
ferredoxin Pdx, comprise therefore a corresponding recognition domain
involved in protein-protein interaction during the general function of
electron transfer.
The efficiency of the cholesterol side chain cleavage is not
significantly changed with Thr-49 substitution mutants. However, no
CYP11A1-dependent enzymatic activity can be detected in
reactions with the deletion mutant T49, which is in agreement with
the finding that no Adx T49
-CYP11A1 complex formation is detectable under the applied conditions. Stopped-flow analysis of the first electron transfer from T49
to CYP11A1 revealed a 6-fold reduced electron flux and indicates a very low binding affinity, which might be
under the detection limit of optical titration experiments, which were
used for estimation of the binding constant. Furthermore, the results
of stopped-flow measurements suggest on the one hand a reduced electron
transfer to CYP11A1 for mutants that contain no alcoholic residue in
position 49, and on the other hand an 3-fold increased electron
transfer after substitution of threonine by the aromatic tyrosine
residue. Interestingly, although the redox potential of mutant T49Y and
the binding constant for CYP11A1 are not changed, the introduced
tyrosine residue seems to form a preferred route for the electron
transfer between Adx and CYP11A1, which is in good agreement with
previous observations that aromatic residues enhance electron transfer
in proteins (45). The results of these experiments indicate that
residue 49 is involved in the electron transfer pathways from Adx to
CYP11A1. This indication is in accordance with the calculated electron
coupling between the [2Fe-2S] cluster and the surface of Adx, which
pointed out a hot spot in the [2Fe-2S] cluster loop containing
residue 49. The experimental and theoretical evidences obtained by
stopped-flow measurements and electron transfer calculations imply that
residue 49 is positioned at the boundary separating the [2Fe-2S]
redox center from the surface of the interacting redox partner protein during complex formation. The nature of the influence of Thr-49 in this
position on the complex formation and electron transfer, which can be a
direct interaction with binding partners or a structure-forming function in the [2Fe-2S] cluster loop, remains unclear and is subject
to further studies.
Taken together, residue Thr-49 of bovine Adx plays an important role in
the stabilization of the hydrogen bond network of the [2Fe-2S]
center. Deletion of this residue markedly decreases the redox potential
of the protein indicating that this residue determines the redox
potential in Adx. Structural rearrangements of the cluster vicinity in
this mutant also led to decreased stability and dramatically reduced
binding affinities of the redox partner proteins AdR and CYP11A1.
Introduction of the smaller alanine and the larger tyrosine in this
position significantly changes the binding constants, providing further
evidence that amino acid Thr-49 is involved in the interaction with the
other proteins in the electron transfer chain. The measured apparent
rate constants for the first electron transfer and the calculated
electron coupling map of the surface of Adx provide evidence for the
participation of the position 49 in the electron pathways between the
iron-sulfur center of Adx and the heme of the cytochrome CYP11A1.
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ACKNOWLEDGEMENTS |
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We thank Wolfgang Reinle and Walter Klose for purifying AdR and CYP11A1, Michael Lisurek for preparing the picture of Adx, and Elena Olkova for calculating the electron coupling values. We greatly acknowledge the technical assistance of Katharina Bompais.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grants Be 1343/1-3 and Be 1343/8-2 and by the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. E-mail: ritabern@rz.uni-sb.de.
Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M007589200
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ABBREVIATIONS |
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The abbreviations used are: Adx, adrenodoxin; AdR, adrenodoxin reductase; CYP11A1, cytochrome P-450 11A1; CYP11B1, cytochrome P-450 11B1; CYP101, cytochrome P-450 cam; HPLC, high performance liquid chromatography; CD, circular dichroism; NMR, nuclear magnetic resonance; Pdx, putidaredoxin.
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