(Received for publication, September 5, 1996, and in revised form, November 27, 1996)
From the An analysis of the electron transfer kinetics
from the reduced [2Fe-2S] center of bovine adrenodoxin and its
mutants to the natural electron acceptors, cytochromes P450scc and
P45011 The key step of different biological reactions such as oxidative
phosphorylation, photosynthesis, respiration, drug metabolism, and many
other processes taking place in living organisms is an electron
transfer. Electron transfer reactions commonly occur between
protein-bound prosthetic groups of the electron donors and acceptors by
protein-protein interaction (1). However, only a little is known about
the geometry of the protein-protein complex required for successful
electron transfer and about the rate-limiting processes.
Bovine adrenodoxin is of particular interest because of its specific
role as an electron carrier associated with steroid hydroxylation reactions. Adrenodoxin is a member of the ferredoxin family of electron-transferring non-heme iron proteins. It is a low molecular mass protein (14 kDa), negatively charged at neutral pH values and
containing the [2Fe-2S] cluster as redox-active group. Adrenodoxin mediates the transfer of electrons from NADPH via adrenodoxin reductase
to the heme iron of mitochondrial cytochromes P450, which are localized
in the inner mitochondrial membrane of bovine adrenal cortex, involved
in the side chain cleavage of cholesterol (CYP11A1),1 the 11 Based on chemical modification studies a charge-pair interaction
mechanism has been proposed between adrenodoxin and its redox partners
(3, 4). Further investigations using site-directed mutagenesis on
recombinant human placenta ferredoxin (5, 6) and CYP11A1 (7) confirmed
the role of electrostatic interactions for the functioning of
adrenodoxin as electron transport protein. The acidic region of
adrenodoxin between residues 72 and 79 was shown as being crucial for
binding to adrenodoxin reductase and cytochrome CYP11A1, implying
common or overlapping binding site(s) on adrenodoxin and its redox
partners. Nevertheless, detailed studies on the single tyrosine 82 residue of adrenodoxin (8) revealed that Tyr-82 seems to play an
essential role in the interaction with CYP11A1 and CYP11B1, but not
with adrenodoxin reductase. Furthermore, the data clearly demonstrated
the different requirements of CYP11A1 and CYP11B1 for recognition of
adrenodoxin, which may be important for allowing the proteins to
discriminate between the oxidized and reduced forms of adrenodoxin to
produce effective electron transfer complexes (12). These observations
were supported by site-directed mutagenesis studies of histidine 56 (9)
and threonine 54 (10) residues and COOH-terminal deletion mutants of
adrenodoxin (11).
Despite much information provided from structural and kinetic studies
of the components of steroid hydroxylase system, there is no definite
model of electron transport from NADPH to the natural acceptor,
cytochrome P450. At present the "shuttle" model (4, 13), a ternary
complex formation of adrenodoxin, adrenodoxin reductase, and cytochrome
P450 (14), and a model suggesting the occurrence of two adrenodoxin
molecules in the electron transport chain (15) are under discussion.
Unfortunately, the tertiary structures of adrenodoxin and its redox
partners, adrenodoxin reductase, CYP11A1, and CYP11B1, which could give
some structural information for understanding the mechanism of electron
transfer at molecular level, have not been solved yet.
In general, for redox partners that associate prior to electron
transfer, the driving force of the reaction depends on both the
reduction potentials of the free species and any differences in the
association free energies for the one-electron reduced complex (16).
Theoretical dependence of electron transfer rate on redox potential has
been shown to be valid in many model systems (17). However, electron
transfer reactions of cytochrome P450 are very complicated and could
meet very different driving forces in different reaction steps (18).
Another point that should be emphasized is that most experimental data
on electron transfer in steroid hydroxylase system were obtained for
CYP11A1. The general conclusions derived from these experiments are not
necessarily true for CYP11B1 if the different physicochemical,
immunochemical, and catalytic characteristics of cytochromes CYP11A1
and CYP11B1 are considered.
To provide more detailed insight into the contribution of electronic
and conformational states to the driving forces for the one-electron
reduced complex formation we performed studies with special attention
to the exclusive role of adrenodoxin. For this purpose a series of
adrenodoxin mutants possessing distinctive structural characteristics
(i.e. redox potential, microenvironment of the iron-sulfur
chromophore, electrostatic parameters, conformational stability) was
used as electron transfer mediators.
The wild type
adrenodoxin and the site-directed mutants were generated as originally
described by Beckert et al. (8, 9). Expression in
Escherichia coli and purification of recombinant proteins
were carried out as reported previously (8). The absorbance ratios
A414/A276 for wild type,
His-56, and Asp-76 mutant adrenodoxin preparations were higher than
0.9. Absorbance ratios of
A414/A276 for the Tyr-82
mutants were higher than 1.0 because of the absence of tyrosine 82. The
concentrations of adrenodoxin proteins were determined using
Adrenodoxin reductase and cytochromes CYP11A1 and CYP11B1 were purified
according to the procedure of Akhrem et al. (20) with slight
modifications. The concentration of adrenodoxin reductase was
determined spectrophotometrically using an extinction coefficient of
11.3 (mM·cm) EPR and CD spectra were recorded as
described previously (8).
Fluorescence emission spectra of protein solutions were taken with an
RF-5001 PC spectrofluorometer by exciting at 270 nm. Measurements were
carried out in 10 mM phosphate buffer, pH 7.4, at room
temperature. Protein concentrations used were in the range 0.03-0.2
mg/ml, where all geometric effects may be eliminated (9, 23).
Redox potentials of the wild type adrenodoxin and the mutants were
measured by the dye photoreduction method with safranin T as indicator
and mediator according to Sligar and Gunsalus (24). Potentials were
quantitated in a 3-ml reaction mixture containing 10 mM
EDTA, 0.3 M glucose, 2.5 µM safranin T, and
100 µl of an oxygen-scavenging system (4 mg/ml glucose oxidase and 2 mg/ml catalase) in 100 mM potassium phosphate buffer, pH
7.5, using the procedure described by Beckert et al. (8).
All data were analyzed according to the Nernst equation.
The assays involving the
measurements of the kinetics of interaction between adrenodoxin and
adrenodoxin reductase using cytochrome c as an electron
acceptor were carried out in the cytochrome c reduction
mixture (1 ml) containing 0.2 µM adrenodoxin reductase, 100 µM horse heart cytochrome c, and
adrenodoxin or adrenodoxin mutants (variable amounts) in 33 mM potassium phosphate buffer, pH 7.4. The reaction was
started by the addition of 120 µM NADPH. The reduction of
cytochrome c was monitored at 550 nm, and the activity was
calculated on the basis of an extinction coefficient of 20 (mM·cm) 11 The cholesterol side chain cleavage activity of the adrenodoxin mutants
was measured in the reconstituted assay system catalyzing the
conversion of cholesterol to pregnenolone according to the procedure of
Sugano et al. (25) with modifications described by Beckert
et al. (8). The incubation mixture contained, in a final
volume of 1 ml, 20 mM potassium phosphate buffer, pH 7.4, 0.3% (w/v) Tween 20, 100 µM cholesterol, 1 µM CYP11A1, 0.5 µM adrenodoxin reductase,
adrenodoxin (variable amounts), and the NADPH-regenerating system. The
reaction was started by adding 60 µM NADPH. After a
10-min incubation at 37 °C, the reaction was stopped by heating to
98 °C. For conversion of steroids into the 3-on-4-ene form, 50 µl
of cholesterol oxidase (0.4 unit) in 20 mM potassium
phosphate buffer, pH 7.4, containing 1% sodium cholate and 100 µM corticosterone as an internal standard was added to
the reaction mixture. The internal standard was necessary because of
the additional isomerase activity of cholesterol oxidase, which makes
the exact determination of the transformed cholesterol amounts
impossible, but not that of pregnenolone formation. After incubation at
37 °C for 10 min, the steroids were extracted with dichloromethane.
The samples were analyzed by reverse phase HPLC with a gradient solvent
system of 70-100% methanol. Cholestenone, progesterone,
corticosterone, and pregnenolone were used as standards.
Titration
was performed according to Kido and Kimura (14). Binding of oxidized
adrenodoxin to the oxidized CYP11A1 was followed spectrophotometrically
by a high spin shift of the P450 heme iron, caused by
adrenodoxin-induced cholesterol binding, using 2.3 µM CYP11A1 in 50 mM potassium phosphate buffer, pH 7.4, with
0.03% Tween 20 and 25 µM cholesterol at room
temperature. Defined amounts of adrenodoxin were added from a stock
solution. Calculation of concentrations of free adrenodoxin was done as
described by Coghlan and Vickery (5).
Kinetic parameters were determined by least squares linear regression
analysis of the Lineweaver-Burk plot. The standard deviations of the
kinetic and binding constants were calculated from data of three or
four separate experiments.
An investigation of the first
electron transfer reaction from adrenodoxin to cytochromes P450 was
carried out using a single channel stopped flow ASVD spectrometer
SX-17MV (Applied Photophysics) according to Lambeth and Kriensgiri (26)
with some modifications. The reduction assays were performed in two
different manners. In a first set of experiments one test tube
contained 0.5 µM adrenodoxin reductase, varying amounts
of adrenodoxin, and 1 µM CYP11A1 in 50 mM
potassium phosphate buffer, pH 7.4, with 0.03% Tween 20 and 25 µM cholesterol, or with 0.01% Tween 80, 1 mM
EDTA, and 25 µM deoxycorticosterone, respectively. The
second test tube contained the reducing component, NADPH (1 mg/ml) in
the respective reaction solution.
The reaction mixtures were treated with carbon monoxide, and an oxygen
scavenging system was added. Electron transfer reaction was followed by
monitoring the absorbance of the ferrous-carbon monoxide-complex at 450 nm, an increase of which was shown to be a precise measurement of
cytochrome P450 reduction by reduced adrenodoxin (27). Electron
transfer rate constants were obtained by a computerized exponential fit
of the data.
In the other case, the reduction assay was essentially similar to the
above mentioned one, with the only exclusion that NADPH was first
incubated with the adrenodoxin/adrenodoxin reductase mixture in the
first test tube, and the oxidized cytochrome P450 (CYP11A1 or CYP11B1)
complexed with the steroid was in the second test tube. The extent to
which the reduction of adrenodoxin by adrenodoxin reductase proceeds
after incubation with NADPH was checked spectrophotometrically using
the method described by Kimura (19).
The main concern of the present study was to relate the essential
structure-function properties of adrenodoxin, which were modified by
site-directed mutagenesis, to the specific aspects of electron transfer
in steroid hydroxylase systems. The surface near to the [2Fe-2S]
cluster of adrenodoxin is of considerable interest since here the
flavoprotein must supply its electrons, and yet it is at this part of
the adrenodoxin surface that the P450 cytochrome must interact. Tyr-82
mutants (i.e. Y82F, Y82S, Y82L) were chosen taking into
consideration two points. On the one hand, mutations of tyrosine 82 were shown to affect differentially interactions of adrenodoxin with
cytochromes CYP11A1 and CYP11B1 (8); and on the other hand, Tyr-82
seems to be located close to the conserved His-56 residue, which is
immediately adjacent to one of the cysteine ligands of the [2Fe-2S]
cluster (9). In addition, adrenodoxin proteins with amino acid
substitutions in position 56 (i.e. H56T, H56Q, and H56R)
were used for our studies, since His-56 has been shown to contribute to
the integrity of the protein region between the iron-sulfur cluster and
the highly conserved acidic region of adrenodoxin, which is important
for binding to both adrenodoxin reductase and P450 (9). Moreover, His-56 seems to play a key role in the stabilization of adrenodoxin structure as became evident from recent calorimetric studies on folding
and stability of adrenodoxin (28).
Taking into account the role of complementary electrostatic
interactions for the association of electron transfer proteins, mutant
D76E of adrenodoxin with a substitution in the highly conserved acidic
region was further selected. This conservative change maintained the
negative charge in position 76 but altered the exact positioning of the
carboxylate moiety potentially inducing disturbances of specific short
range protein-protein interactions as was shown for a heterologous
system consisting of human placenta ferredoxin, bovine adrenodoxin
reductase, and CYP11A1 (6).
The wild type and the mutants were expressed in similar yields as
holoprotein and exhibited absorption CD and EPR spectra characteristic
for the correctly assembled iron-sulfur cluster, indicating that
incorporation of the cluster was not disturbed upon amino acid
replacements (data not shown).
The redox potentials of the mutant proteins provide more detailed
insight into the local environment of the iron-sulfur center in these
proteins (29). The effect of amino acid replacement near the cluster is
sharply defined in the case of His-56 substitution. Replacement of
His-56 by Gln, Arg, or Thr led to marked changes in the redox
potentials of the mutants being 28, 65, and 66 mV, respectively, lower
compared with that of the wild type (Table I). These
changes demonstrate that histidine in position 56 affects the ligand
field around the iron-sulfur cluster. Some lowering (8 mV) in the redox
potential was estimated for mutant Y82S (
Redox potentials of the adrenodoxin mutants
Max-Delbrück-Centrum für
Molekulare Medizin,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
, is the primary focus of this paper. A series of mutant
proteins with distinctive structural parameters such as redox
potential, microenvironment of the iron-sulfur cluster, electrostatic
properties, and conformational stability was used to provide more
detailed insight into the contribution of the electronic and
conformational states of adrenodoxin to the driving forces of the
complex formation of reduced adrenodoxin with cytochromes P450scc and
P45011
and electron transfer. The apparent rate constants of P450scc
reduction were generally proportional to the adrenodoxin redox
potential under conditions in which the protein-protein interactions
were not affected. However, the effect of redox potential differences
was shown to be masked by structural and electrostatic effects. In
contrast, no correlation of the reduction rates of P45011
with the
redox potential of adrenodoxin mutants was found. Compared with the
interaction with P450scc, however, the hydrophobic protein region
between the iron-sulfur cluster and the acidic site on the surface of
adrenodoxin seems to play an important role for precise complementarity
in the tightly associated complex with P45011
.
-hydroxylation of
11-deoxycorticosterone and 11-deoxycortisol, and the production of
aldosterone (CYP11B1). The flavin prosthetic group of adrenodoxin
reductase accepts two electrons from NADPH and contributes one electron
to adrenodoxin. Reduced adrenodoxin in turn transfers an electron to
either ferric or oxygen-bound P450 species. Both side chain cleavage
and 11
-hydroxylation utilize molecular oxygen and require two
electrons per oxidation (six electrons in total for side chain
cleavage) (2). Recognition of cytochromes P450 and the corresponding
electron donor is a significant prerequisite of the catalytic
cycle.
Preparation and Quantitation of Proteins
414 = 9.8 (mM·cm)
1 (19).
1 at 450 nm (21). The
concentrations of cytochromes P450 were estimated according to Omura
and Sato (22).
1 at 550 nm for cytochrome
c.
-Hydroxylation activity measurements were performed as described
previously (8). The reaction mixture (0.5 ml) consisted of CYP11B1 (0.4 µM), adrenodoxin reductase (0.4 µM),
adrenodoxin or adrenodoxin mutants (variable amounts),
deoxycorticosterone (100 µM), and the NADPH-regenerating
system (64 µM glucose 6-phosphoric acid disodium salt and
2 units of glucose-6-phosphate dehydrogenase) in 50 mM
potassium phosphate buffer, pH 7.4, with 1 mM EDTA, 0.1 mM dithioerythritol, 0.01% (w/v) Tween 80. The conversion
of deoxycorticosterone to corticosterone was carried out at 37 °C
for 10 min after initiation of the reaction with NADPH. The reaction
was stopped with dichloromethane, which simultaneously extracts the
steroids. The extracted steroids were then dried under a nitrogen
stream and analyzed by reverse phase HPLC with 70% methanol as a
solvent at a flow rate of 1 ml/min. Corticosterone and
deoxycorticosterone were used as external standards.
282 mV), indicating that
introduction of the small polar serine residue instead of the
hydrophobic tyrosine residue could produce conformational changes of
the cluster environment since the microenvironment of the iron-sulfur
chromophore of native adrenodoxin is known to be largely hydrophobic
(30). Redox potentials obtained for other Tyr-82 mutant proteins, in
which tyrosine was changed to the hydrophobic phenylalanine or leucine
(i.e. Y82F and Y82L), were found to be not significantly
different from those of the wild type (
274 mV). The redox potential
of the D76E mutant remains unchanged, confirming the previous
suggestion that residues in this position are most likely not located
in the immediate vicinity of the cluster (6).
Adrenodoxin
Redox potential
mV
WTa
274
Y82F
274
Y82S
282
Y82L
274
H56Q
302
H56T
340
H56R
339
D76E
274
a
WT, wild type.
Theoretical analysis of electron transport reactions, based on the outer sphere mechanism, predicts that a relationship should exist between the rate constant of the electron transfer and the redox potential difference between donor and acceptor (17). Indeed, a clear correlation was shown to exist between the rate constant for the reduction and the redox potential of the reactants in a structurally homologous series of electron transfer proteins (31). The regulation of the CYP11A1 redox potential by substrates and the influence of these changes on the rate of the electron transfer from reduced adrenodoxin to cytochrome have been investigated by Lambeth and Kriensgiri (26).
Here we have investigated in more detail the relationship among
structural properties of adrenodoxin mutants and the rates of
cytochromes CYP11A1 and CYP11B1 reduction using the stopped flow
technique. Electron transfer rate constants were obtained using
increased concentrations of the wild type and the mutant adrenodoxin
proteins. The assay for CYP11A1 reduction was performed in two
different manners. In the first case, reduction of the heme iron was
measured after the reaction was started with NADPH as reducing
component. In this regime a certain time is needed for interaction of
oxidized adrenodoxin with reduced adrenodoxin reductase and the
electron transfer from adrenodoxin reductase to adrenodoxin followed by
electron transfer from adrenodoxin to cytochrome. The kinetics of
cytochrome reduction was first order at all ratios of adrenodoxin to
cytochrome CYP11A1. Fig. 1A shows plots of
apparent first order rate constants (kapp)
versus the adrenodoxin concentration. In contrast to
observations of Lambeth and Kriensgiri (26), the data demonstrate a
clear increase in the CYP11A1 reduction rates by increasing
concentrations of adrenodoxin with an apparent saturation at a ratio of
approximately 10:1 (adrenodoxin/cytochrome). Since adrenodoxin and
cytochrome P450 associate in the electron transfer complex prior to
electron exchange, intracomplex electron transfer seems to become the
rate-limiting step at high concentrations of adrenodoxin. The lack of
concentration dependence of the reduction rates observed previously
(26) could be due to the presence of dithionite as reducing component
in the stopped flow assay. As evident from Fig. 1A, the
mutant proteins Y82S, H56R, and D76E exhibited significantly lower
kapp values than wild type adrenodoxin. All
other mutants reduced CYP11A1 with essentially the same rate constant
as does wild type adrenodoxin (Fig. 1A).
The second set of experiments was performed to avoid a possible
contribution of adrenodoxin reduction or oxidation by adrenodoxin reductase to the measured rate of electron transfer between adrenodoxin and CYP11A1. For this purpose, adrenodoxin was preincubated with adrenodoxin reductase and NADPH in the first test tube. The other test
tube contained oxidized cytochrome CYP11A1. The molar ratios of
proteins were identical to those for the preceding stopped flow assays.
Analyses of the adrenodoxin absorption spectra after incubation with
adrenodoxin reductase and NADPH in the oxygen-free solution indicated
that at the start point of the stopped flow measurement, when the
mixture containing adrenodoxin reductase, NADPH, and adrenodoxin or its
mutants was mixed with oxidized cytochrome, the wild type adrenodoxin
as well as the mutant proteins were fully reduced by adrenodoxin
reductase (data not shown), thus being appropriately dissociated from
the flavoprotein (32). In this case the rate constants of electron
transfer from the reduced adrenodoxin to the oxidized cytochrome must
be totally adrenodoxin-dependent since the flavin- to
iron-sulfur electron transfer is not rate-limiting under these
conditions. As evident from Fig. 1B, the wild type
adrenodoxin and the mutants, which have unchanged redox potentials
(Table I) and no significantly changed affinity to adrenodoxin
reductase and to CYP11A1 (Tables II and
III) (i.e. Y82F, Y82L), revealed about the
same values of the reduction rates as in the preceding stopped flow
assay. This observation indicates that at least for these adrenodoxin
proteins association and dissociation of the complexes of adrenodoxin
with adrenodoxin reductase as well as a reduction of adrenodoxin occur more rapidly than the measured overall rate of electron transfer. In
contrast, the reduction rates of CYP11A1 were accelerated nearly 2.3-fold using H56T or H56Q mutants. A comparison of data presented in
Fig. 1, A and B, allows the conclusion that in
the case of the reaction start by NADPH (Fig. 1A) the
obtained values of electron transfer rates to CYP11A1 for these mutants
reflect their less favorable electron acceptance from adrenodoxin
reductase. This led us to suppose that for defined mutants the
reduction of adrenodoxin by adrenodoxin reductase is influencing the
overall electron transfer rate from adrenodoxin to cytochrome under
certain conditions. Binding studies using optical difference
spectroscopy revealed that oxidized H56Q exhibits the same affinity for
oxidized CYP11A1, whereas H56T has a slight increase in
Kd value (Table III). Since the association energies
appear to be similar for the interaction of oxidized and reduced
adrenodoxin with the oxidized CYP11A1·substrate complex, a
correlation of the rate constants for the reduction with the redox
potential for these mutants becomes apparent (Fig. 2).
As indicated above, the reduction potentials of the H56Q and H56T
mutants are significantly lowered compared with that of the wild type
(274 mV), being
302 and
340 mV, respectively, thus making the
electron donation to the heme iron of CYP11A1 more favorable. The rate
constants for the adrenodoxin to CYP11A1 electron transfer are
decreased significantly in the case of mutant proteins Y82S and D76E
under reaction conditions of the second variant of stopped flow
experiments (Fig. 1B). Since these mutations do not
appreciably influence redox potential values of the adrenodoxin
proteins, the observed decrease of the rate constants seems to reflect
a strong influence of these mutations on complex formation with
CYP11A1. Particular emphasis must be given to the behavior of mutant
H56R. This protein possesses nearly the same redox potential as does
H56T (
339 and
340 mV, respectively). However, the rate constants
obtained for mutant H56R are considerably lower than those for mutant
H56T. This can be due to the proximity of the voluminous positively
charged arginine residue to the highly acidic binding region of
adrenodoxin. Thus, H56R and H56T are excellent examples for the
interplay of factors that are known from the theory to influence the
rates of electron transfer reactions between proteins, i.e.
the difference in redox potential between reactants and electrostatic
effects of protein-protein interaction. It is evident from Fig. 2 that
in a series of structurally homologous proteins, in which the
interaction with CYP11A1 was not altered significantly upon single
amino acid substitution (i.e. Y82F, Y82L, H56Q, H56T) the
redox potential difference was the dominant factor in determining
electron transfer rate. Approximately linear correlation
(r = 0.86) between redox potentials and rate constants for reduction could be shown in this protein group. In contrast, for
the mutant proteins Y82S, H56R, and D76E structural and electrostatic effects become much more important, masking the effect of redox potential differences on electron transfer rates.
|
|
An investigation of the first electron transfer from adrenodoxin to
cytochrome CYP11B1 catalyzing 11 hydroxylation of
deoxycorticosterone was carried out taking into consideration stopped
flow data obtained for CYP11A1. For these measurements adrenodoxin was
incubated with NADPH and adrenodoxin reductase prior to mixing with the oxidized CYP11B1·substrate complex. As for CYP11A1 reduction we obtained a clear dependence of the first-order rate constants of
electron transfer on adrenodoxin concentration (Fig. 3).
However, the kapp values were lowered nearly
2-fold using CYP11B1 at the same stoichiometry of the reaction
components, coinciding with earlier suggestions about a less effective
transfer of reducing equivalents to CYP11B1 compared with CYP11A1 (33).
Additionally, as shown in Fig. 4, no correlation of the
reduction rates of CYP11B1 on the redox potential of the adrenodoxin
mutants was found. Moreover, the reduction rates of the D76E mutant are
approximately the same compared with those of the wild type, whereas
they are 5-fold lower in the reduction assay with CYP11A1. In addition,
CYP11B1, although not sensitive to the replacement D76E, seems to be
more sensitive than CYP11A1 to the properties of the amino acids
substituting positions 56 and 82. Analyzing the data shown in Fig. 3
revealed that interaction between CYP11B1 and adrenodoxin is
substantially of hydrophobic nature. The hydrophobic protein region
between the iron-sulfur cluster and the acidic site on the surface of the adrenodoxin seems to play an important role for the precise complementarity of the tightly associated complex with CYP11B1.
Successively, we have examined comparatively how the kinetics of the first electron transfer is related to catalytic and binding properties of the adrenodoxin mutants in cytochrome c reduction and CYP11A1- or CYP11B1-dependent substrate conversion. These studies have demonstrated that amino acid substitutions that alter the electrostatic properties of adrenodoxin (i.e. Y82S, H56R, D76E) decreased the activity and affinity of these mutants for both adrenodoxin reductase and CYP11A1 as evidenced by raised Km and Kd values (Tables II and III). As expected, the effect of imbalance of the electrostatic interactions is most pronounced for mutant D76E, exhibiting a more than 10-fold decrease in the affinity to adrenodoxin reductase as measured by a respective increase in the Km value of the cytochrome c reduction (Table II) Moreover, although adrenodoxin mutant D76E exhibits a 10-fold increase in the Kd value for CYP11A1 as evident from the spectral binding studies (Table III), the Km value for this mutant in the CYP11A1 substrate conversion assay reveals under our experimental conditions only a 2.6-fold increase compared with the wild type (Table II). Of particular interest is the similarity of the electrostatic effects caused by H56R and D76E replacements concerning adrenodoxin interaction with adrenodoxin reductase and CYP11A1. Replacement of His-56 with arginine residue results in ~5-fold decreases in binding affinities for both adrenodoxin reductase and CYP11A1 (Tables II and III).
Analysis of the enzymatic activity of adrenodoxin mutants with respect to CYP11B1-dependent substrate conversion suggests differences in the binding requirements of this protein compared with CYP11A1. Close examination of the Km values indicates that the adrenodoxin mutants with charged or polar residues in positions 56 and 82 (i.e. H56Q, H56R, and Y82S) show decreased affinities to the CYP11B1. Interestingly, increases of the Km values for mutants H56R, H56Q, and H56T are greater than that of mutant D76E (Table II). On the other hand, replacing Tyr-82 with the more hydrophobic resides leucine or phenylalanine increased the ability of these mutants to interact with CYP11B1 as evidenced in 2.5-5-fold lower Km values.
However, the interpretation of the Km values obtained for CYP11A1 and CYP11B1 becomes more complicated since the turnover of these systems, being totally adrenodoxin-dependent, could become dependent on the interaction between adrenodoxin and adrenodoxin reductase, at least for the mutants possessing affected affinity to adrenodoxin reductase. As was clearly demonstrated in our stopped flow investigations, such flavin- to iron-sulfur electron transfer limitation would mask the measured kinetic constants for cytochrome reduction.
The Vmax values remain unchanged for all mutants in cytochrome c reduction and cholesterol or deoxycorticosterone conversion assays (data not shown), demonstrating that the efficiency of substrate conversion at infinite adrenodoxin concentrations is not influenced by amino acid substitution in positions 56, 82, or 76. Since the second electron transfer appears to be rate-determining for P450-dependent mixed function oxidations (13), it seems likely that the kinetic constants are primarily determined by second electron transfer.
In conclusion, it has been shown that a linear correlation between the redox potential of various adrenodoxin mutants and the apparent rate constant of CYP11A1 reduction can be observed. The correlation does not hold true for mutants changing the electrostatics and/or structure of the interaction site (i.e. H56R, D76E). Such a correlation could also not be obtained for CYP11B1 reduction. Our data led us to conclude that for adrenodoxin/CYP11B1 interaction hydrophobic properties of the interaction site are more important.
Interestingly, in some cases the rate of the first electron transfer does not reflect the rate of the efficiency of substrate conversion. This indicates that either the first and second electron transfers are differentially regulated (implying different requirements for the adrenodoxin·P450 electron transfer complex) or that another step (e.g. product release) is rate-limiting in these systems.
We thank Dr. R. Wessel for help with the stopped flow measurements and C. Jaeger for highly skilled technical assistance. V. B. expresses deep gratitude to Dr. A. Lapko for stimulating discussion.