From the Department of Structural and Functional
Biology, University of Insubria, via J.H. Dunant 3, 21100 Varese, Italy
and the § Faculty of Biology, University of Konstanz, P. O. Box 5560-M644, D-78434 Konstanz, Germany
Received for publication, December 5, 2000, and in revised form, February 13, 2001
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ABSTRACT |
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Brevibacterium sterolicum possesses
two forms of cholesterol oxidase, one containing noncovalently bound
FAD, the second containing a FAD covalently linked to His69
of the protein backbone. The functional role of the histidyl-FAD bond
in the latter cholesterol oxidase was addressed by studying the
properties of the H69A mutant in which the FAD is bound tightly, but
not covalently, and by comparison with native enzyme. The mutant
retains catalytic activity, but with a turnover rate decreased 35-fold;
the isomerization step of the intermediate 3-ketosteroid to the final
product is also preserved. Stabilization of the flavin semiquinone and
binding of sulfite are markedly decreased, this correlates with a lower
midpoint redox potential ( The flavoprotein cholesterol oxidase
(CO)1 (EC 1.1.3.6) is an
alcohol dehydrogenase/oxidase that catalyzes the dehydrogenation of
C(3)-OH of the cholestan system to yield the corresponding carbonyl
product (Scheme 1). During the reductive half-reaction, the oxidized
FAD accepts a hydride from the alcohol, and in the ensuing oxidative
half-reaction, the reduced flavin transfers the redox equivalents to
molecular oxygen, which is the final acceptor. COs are a group of
enzymes isolated (and frequently secreted) from various microorganisms,
they are characterized by a broad substrate specificity within the
cholestan family. The first three-dimensional structure of a CO was
that of the enzyme from Brevibacterium sp., which contains a
noncovalently linked FAD (1); the structure of the CO from
Streptomyces hygroscopicus (SCO) has recently been reported
(2). Both enzymes possess the classical Rossman fold for dinucleotide
binding found in many flavin-dependent oxidases (3) and
belong to the glucose-methanol-choline oxidoreductase family
(4).
A second CO was isolated from a strain reported to be a
Brevibacterium sterolicum (BCO) (5). Intriguingly, it has a
different primary sequence, it contains a FAD covalently linked to N(1) of His69, and its three-dimensional structure belongs to a
different family compared with the two COs mentioned
above.2 A similar situation
was also reported in the two related enzymes D- and
L-nicotinic acid oxidase from Arthrobacter
oxidans: one contains covalently linked FAD and is specific for
the substrate D-form (6); the second acts on the
L-form and does not possess a covalent flavin. Some
properties of the COs from S. hygroscopicus (containing
noncovalently linked FAD) and from a recombinant protein from B. sterolicum (with covalently linked FAD) were described and
compared recently (7). The effects of organic solvents, surfactants,
and ionic strength on CO activity have been investigated (8). With only
the exception of glucose oxidase, CO is the most widely used enzyme in
clinical laboratories (for a recent and exhaustive review on CO
applications in analytical chemistry, see Ref. 9).
BCO catalysis proceeds via a ping-pong mechanism, whereas SCO follows a
sequential pathway (10). Although the two enzymes have similar rates of
flavin reduction (~230 s Up to now, more than 30 flavoenzymes have been reported to contain
flavin covalently linked to a histidine, cysteine, or tyrosine residue
of the polypeptide chain (6, 13). The BCO used in the present work
contains covalent flavin and is structurally distinct from the
previously studied COs because it belongs to the subfamily of
vanillyl-alcohol oxidase, that contains a fold proposed to favor
covalent flavinylation (14). Although the existence of covalent
flavin-protein linkages has been known for many years, only recently
has its mechanism of formation emerged and its functions been proposed
(13): (i) it allows saturation of the active site with the cofactor
(particularly favorable for flavoenzymes that are localized in a
flavin-deficient environment); (ii) it modifies the redox properties;
(iii) it modulates substrate specificity; (iv) it protects the coenzyme
from modification (i.e. hydroxylation) and inactivation; (v)
it facilitates the electron transfer to another coenzyme; and (vi) it
induces structural benefits such as improved protein stability.
To elucidate the function of covalent flavin linkage in BCO, we have
studied a mutant in which the exchange His69 Materials and Enzymes--
Cholesterol and ThesitTM
(dodecyl poly(ethyleneglycol ether)n, n = 9-10) were purchased from Roche Molecular Biochemicals. All other
reagents were of the highest commercially available purity. Wild-type
and H69A recombinant B. sterolicum CO, from Escherichia coli, were obtained from Roche Molecular
Biochemicals.
Enzymatic Activity--
Cholesterol oxidase activity was assayed
at 25 °C monitoring H2O2 production at 440 nm ( Absorption and Fluorescence Measurements--
UV-visible
absorption spectra were recorded with an Uvikon 930 spectrophotometer
(Kontron Instruments) in 100 mM potassium phosphate buffer,
pH 7.5, at 25 °C. Extinction coefficients were determined by
measuring the change in absorbance by heat denaturation (boiling 5 min
in the dark). An extinction coefficient of 11.3 mM
The dissociation constant of the apoprotein-coenzyme complex was
calculated using the Stinson equation (17) and assuming a simple 1:1
equilibrium (PF Enzyme Stability--
To determine the stability in solution of
wild-type and H69A BCO, enzyme samples were incubated at 25 °C and
at 0.1 mg/ml final protein concentration in 50 mM potassium
phosphate buffer, pH 7.5. To assess the effect of propan-2-ol on enzyme
stability, the samples (0.1 mg of protein/ml) were incubated in the
presence of 0-60% (v/v) propan-2-ol. In all cases, aliquots were
retrieved periodically and assayed for cholesterol oxidase activity
using the H2O2 peroxidase method.
Redox Potentials--
Redox potentials for the
EFlox/EFlseq and
EFlseq/EFlred couples of CO were determined
using the dye equilibration (18) method with the xanthine/xanthine
oxidase reduction system at 15 °C (19). An anaerobic cuvette
containing Preparation of Apoprotein--
The apoprotein of H69A BCO was
prepared by overnight dialysis at 4 °C against 250 mM
Tris buffer, pH 8.5, containing 2.5 M KBr, 20% glycerol,
0.3 mM EDTA, and 5 mM 2-mercaptoethanol. The sample was then desalted by gel permeation chromatography on a PD-10
column (Sephadex G-25) equilibrated with 100 mM potassium phosphate buffer, pH 7.5.
Spectral Properties and Reaction with Substrate--
In comparison
to wild-type BCO, the visible spectrum of the H69A mutant exhibits some
differences both in the oxidized and reduced states (Fig.
1). In addition to the main band in the
visible, oxidized H69A BCO has a shoulder at
The pKa value for the deprotonation of the flavin
N(3)H position was estimated from the pH dependence of the visible spectrum of the oxidized enzymes. For wild-type BCO the
pKa is Stability--
The stability of BCO in solution was studied by
assessing the activity of each enzyme form at 25 °C in the presence
of 50 mM potassium phosphate buffer, pH 7.5, and as a
function of time. Both wild-type and H69A cholesterol oxidases
maintained more than 90% of the initial activity after 300 min at
25 °C (Fig. 2). The effect of organic
solvents, surfactants, and ionic strength on CO catalysis is
fundamental, since these factors affect the solubility of steroid
substrates (very low in aqueous media), the micellar composition of the
system and the enzyme activity (8, 10). Thus, we investigated the
effect of propan-2-ol in the final concentration range 0-60% (v/v) on
the time dependent activity of the two enzymes (shown in Fig. 2).
Somewhat surprisingly, the (in)stability of the H69A mutant is quite
comparable to that of wild-type BCO (Fig. 2), the main difference being
the Kinetic Properties--
The reaction of H69A BCO with cholesterol
was studied using the H2O2 assay (7, 8, 10) and
the EMTN method (15) under standard conditions (50 mM
potassium phosphate buffer, pH 7.5, 1% Thesit, 1 or 10% propan-2-ol).
With the first assay, and at a fixed (21%) O2
concentration, a strong decrease in catalytic activity is evident for
the H69A mutant: kcat is reduced 30-fold and
Km increased 6-fold compared with the wild-type BCO (Table II). Although the
kcat values are quite similar at 1 and 10%
(v/v) propan-2-ol, the increase in the alcohol concentration resulted
in a sharp increase in the Km value for cholesterol and with both wild-type and H69A COs. As wild-type BCO, the mutant catalyzes the isomerization reaction, i.e. the conversion of
5-cholesten-3-one, the assumed intermediate, into 4-cholesten-3-one,
the final product (Scheme 1). Comparison
of the rate of isomerization with those for oxidation of cholesterol
(Table II) show that the
The kinetic mechanism of BCO with cholesterol as the substrate was
recently studied by a combination of steady state and pre-steady state
approaches (10). Based on these results, BCO was proposed to work via a
ping-pong (binary complex) mechanism. The steady state kinetic behavior
of H69A BCO was analogously studied using the EMTN method and
cholesterol as the substrate. The oxidized enzyme was mixed aerobically
with cholesterol and the change in flavin absorption monitored at 446 nm. A very rapid decrease in absorption was observed, amounting to
Preparation of H69A BCO Apoprotein and Reconstitution with
FAD--
Several methods were explored in order to obtain the
apoenzyme of H69A BCO. We established that mild dialysis in the
presence of 2.5 M KBr and 20% glycerol produces good
yields and a preparation that could be reactivated with a >80%
recovery (the monovalent bromide anion competes with coenzyme,
weakening the binding of flavin to apoproteins, whereas glycerol favors
protein stabilization). The properties of the reconstituted holoenzyme
are nearly identical to those of the starting H69A BCO with respect to
spectral properties and specific activity (~0.8 units/mg of protein).
Binding of FAD to the apoprotein was measured by following the
quenching of protein fluorescence (emission at 340 nm), as well as by
following the increase in flavin fluorescence (emission at 530 nm)
during the titration of 0.3 µM apoprotein with FAD. A
Kd value of 3.7 × 10
Formation of active H69A holoenzyme during the incubation of apoprotein
with excess FAD at 15 °C under the experimental conditions used for
the fluorometric analysis was followed using the
o-dianisidine activity assay. The increase in absorbance at
440 nm shows only a short lag, activity being fully regained during the
first phase as defined by the fluorescence decrease experiments. The
nature of the second rearrangement is unknown.
Redox Potentials--
A preliminary indication of a modification
of the redox properties of H69A BCO arises from the sulfite reactivity.
Addition of sulfite to oxidized wild-type BCO results in essentially
complete bleaching of the oxidized flavin spectrum (7). Under the same conditions, the reaction of the H69A mutant is very slow and is not
completed after 41 h, reflecting a Kd
Therefore, the redox potential of the H69A BCO was measured in order to
assess the effect of the mutation on the thermodynamic properties of
the flavin. When the xanthine oxidase-mediated reduction of H69A BCO
was monitored in the absence of a reference dye, the percentage of
semiquinone formed during the reduction (Fig.
4) was close to zero (~70% with
wild-type BCO), i.e. the values of the potentials for
transfer of each single electron were similar. The estimated separation
between the potentials is Properties of (8Cl-FAD)-H69A BCO--
8-Chloro-FAD was chosen to
reconstitute H69A apoprotein because of its higher potential with
respect to unmodified FAD (Em =
The redox potential of (8Cl-FAD)-H69A BCO is The present results demonstrate that covalent flavinylation is not
a prerequisite for CO protein folding and for efficient binding of FAD.
The latter could be removed reversibly as previously reported for other
flavoproteins (24). In the binding experiments a Kd
Our results are compatible with the concept that covalent flavinylation
as such is not a requirement for catalysis. The observed effects can be
attributed largely to the decrease in the redox potential.
From a kinetic point of view based on EMTN measurements, wild-type and
H69A BCO function by a binary complex mechanism which is similar to
that reported previously for intact enzyme (10), the main difference
being a Interestingly, the Em value for H69A BCO ( Effects similar to those described here have been reported recently for
vanillyl alcohol oxidase, where an analogous H422A mutation and the
consequent absence of covalent flavin linkage lead to a ~120 mV more
negative Em potential (23). Importantly, with
vanillyl alcohol oxidase the three-dimensional structural data suggest
that the effect on the midpoint redox potential is not due to
structural alterations and that the flavin covalent link does not
induce specific structural features. We anticipate a similar
verification from the results of the study of the crystal structure of
BCO and H69A BCO. The main difference between BCO and vanillyl oxidase
is, thus, the great stabilization of the semiquinone form in vanillyl
alcohol oxidase (23), whereas the two-electron transfer is
thermodynamically favored with H69A BCO. The thermodynamic effects
induced by the covalent flavin linkage can be mimicked successfully
using 8Cl-FAD, an analogue with a 65-mV higher potential than that of
normal FAD. This is also manifested in the sulfite reactivity. The
holoenzyme of H69A BCO reconstituted with the "high potential"
8-chloro-FAD analogue shows a In conclusion, it appears that the flavin 8204 mV compared with
101 mV for
wild-type). Reconstitution with 8-chloro-FAD led to a holoenzyme form
of H69A cholesterol oxidase with a midpoint redox potential of
160
mV. In this enzyme form, flavin semiquinone is newly stabilized, and a
3.5-fold activity increase is observed, this mimicking the
thermodynamic effects induced by the covalent flavin linkage. It is
concluded that the flavin 8
-linkage to a (N1)histidine is a pivotal
factor in the modulation of the redox properties of this cholesterol
oxidase to increase its oxidative power.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 in the presence of 1% Thesit
and 10% propan-2-ol), the turnover number at infinite oxygen and
cholesterol concentrations is higher for SCO than for BCO (202 s
1 and 105 s
1, respectively). Alcohols can
be used as detergents and are useful for solubilizing steroids such as
cholesterol. The effect of propan-2-ol on CO consists in an increase of
enzyme activity with increasing concentration of the alcohol up to 10%
(v/v) (8). This has been attributed to the effect of the alcohol on the
availability of substrate that is incorporated into micelles and to the
decrease of the critical micelle concentration for cholesterol (8). While SCO is slowly reduced by propan-2-ol under anaerobic conditions with 1.3 M propan-2-ol (half-time
86 min at 25 °C),
BCO does not react under similar conditions (10). The isomerization
reaction (Scheme 1) is efficiently catalyzed by the oxidized form of
both enzymes and is never rate-limiting in catalysis. In the last few years, the reaction mechanism of CO has also received some attention (11), and studies based on directed mutagenesis have been conducted (12).
Ala
prevents formation of the histidyl-FAD bond. This was also spurred on
by the expected results of the study of the three-dimensional structure
of BCO. Comparison of the structure of the present BCO with the crystal
structure of the noncovalent CO from B. sterolicum, particularly in light of the data presented here, is expected to expand
our understanding of the role of the flavin covalent attachment in
flavoprotein oxidases.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
440 = 13 mM
1
cm
1) in an enzyme-coupled assay with 10 µg/ml
horseradish peroxidase and 16 mg/ml o-dianisidine as
previously described (7, 8, 10). The isomerization reaction (Scheme 1)
is followed by monitoring the production of 4-cholesten-3-one from
5-cholesten-3-one spectrophotometrically at 240 nm
(
240 = 15.5 mM
1
cm
1) due to the formation of two conjugated double bonds.
Another procedure used to assay the CO activity was the
enzyme-monitored turnover (EMTN) method of Gibson et al.
(15), in which the enzyme was mixed anaerobically with substrate in a
stopped-flow instrument and the absorption change was monitored at 446 nm to follow reaction progress. At this wavelength the conversion of
oxidized into reduced, enzyme bound flavin is detected. All
concentrations mentioned in these experiments are those after mixing,
i.e. at 1:1 dilution. Rapid reactions were routinely
recorded in the 300-700 nm wavelength range with an acquisition time
of 0.8 ms/spectrum and resolution of 1 nm, as detailed in Ref. 10.
Enzyme activity was assayed in 50 or 100 mM potassium
phosphate buffer, pH 7.5, containing 1% (v/v) Thesit and 1% or 10%
(v/v) propan-2-ol. EMTN data were analyzed using traces at 446 nm
according to the method of Gibson et al. (15) and using
KaleidaGraphTM (Synergy Software).
1 cm
1 at 445 nm for free FAD
and one of 10.6 mM
1 cm
1 at 445 nm for free 8Cl-FAD (8-chloro-FAD) were used. Photoreduction experiments were carried out at 15 °C using a cuvette containing
7.5 µM enzyme, 5 mM EDTA, and 0.5 µM 5-deazaflavin which had been rendered anaerobic by
subjecting it to alternative cycles of vacuum and O2-free
argon. The enzyme was photoirradiated (7, 16) and the progress of the
reaction was followed spectrophotometrically. Flavin and protein
fluorescence measurements were carried out in a Jasco FP-750
spectrofluorometer in 100 mM potassium phosphate buffer, pH
7.5, at 15 °C. Protein fluorescence emission was monitored at 340 nm
(
exc = 285 nm) and flavin fluorescence emission at 530 nm (
exc = 450 nm), using an excitation slits = 5 nm
and an emission slit = 10 nm. The fluorescence data were analyzed
using the Spectra Analysis Program (Jasco Corp.).
where a, fractional saturation of the total
concentration of binding sites; Kd, dissociation
constant of the enzyme-ligand complex; [L0], total ligand
concentration; and [E0], total enzyme concentration.
(Eq. 1)
12 µM enzyme, 0.2 mM xanthine, 5 µM benzyl viologen, and 1-10 µM of the
appropriate dye was purged of oxygen, and the reaction was initiated by
addition of 10 nM xanthine oxidase. The reaction was
measured spectrophotometrically until completion, typically 3-4 h.
Data were analyzed as described by Minnaert (18). The amount of
oxidized and reduced dye was determined at a wavelength at which the
enzyme shows no absorbance (>500 nm), and the amount of oxidized and
reduced enzyme was determined at an isosbestic point for the dye.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
395 nm and lacks the
band in the near UV typical of wild-type BCO. The spectrum of the
mutant also exhibits a substantial increase in the extinction
coefficient of the band at 444 nm (
= 16.07 and 13.4 mM
1 cm
1 for H69A and wild-type
BCO, respectively). Anaerobic addition of excess cholesterol results in
full flavin reduction (Fig. 1), demonstrating that the noncovalently
bound FAD mutant is competent in catalysis. The reduced form of H69A
BCO is spectrally different from wild-type BCO in that the 376 nm band
is replaced by a shoulder at
389 nm (Fig. 1). An anaerobic titration
of H69A BCO with cholesterol is depicted in Fig. 1. The intercept of
the initial slope of the titration curve with the maximal, observed
absorbance change at 445 nm indicates a stoichiometry
1 for the
reaction with substrate (16.3 nmol of enzyme and 18 nmol of substrate,
inset of Fig. 1). Similar results were obtained with
wild-type and (8Cl-FAD)-H69A BCO (not shown). During the anaerobic
titration with H69A BCO, a small absorbance at
550 nm is evident
that might be attributed to formation of a small quantity of anionic
radical. Stabilization of the anionic semiquinone is typical for
cholesterol oxidase (7) and for the family of flavoprotein oxidases
(20). The amount of semiquinone (EFlseq) formed by
wild-type BCO upon anaerobic photoreduction is
70% of the
extrapolated, possible maximum (Fig. 1). With the mutant this is
reduced to
5% (Table I). Mutant and
wild-type BCO display similar fluorescence emission spectra with maxima
at 535 nm (
exc = 450 nm) and very low quantum yields (
3% of the fluorescence of free FAD with wild-type BCO, and
10% with H69A BCO).
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Fig. 1.
Spectral properties of wild-type, H69A, and
(8Cl-FAD)-H69A mutant forms of BCO and anaerobic reaction of H69A BCO
with cholesterol. Top panel: solid line,
oxidized wild-type BCO in 100 mM potassium phosphate
buffer, pH 7.5, at 15 °C; long dashed line, semiquinone
form generated by photoirradiation in the presence of 5 mM
EDTA and 0.5 µM 5-deazaflavin; dashed line,
fully reduced enzyme obtained from anaerobic reaction with 50 µM cholesterol. Central panel: solid
line, oxidized H69A BCO. This sample (1.8 ml of solution of 9.05 µM H69A BCO, corresponding to 16.3 nmol of enzyme in 100 mM potassium phosphate buffer, pH 7.5) was anaerobically
titrated with small additions of a cholesterol solution (0.375 mM in 50 mM potassium phosphate buffer, pH 7.5, containing 1% Thesit and 1% propan-2-ol), and at 15 °C. Selected
spectra (dotted line) are shown for the following total
concentrations of cholesterol (from top at 445 nm): 3.375, 5.25, 8.625, 10.5, 12.375, 19.5, and 38.25 nmol. Inset, dependence of the
absorbance at 445 nm (upon correction for dilution) on the cholesterol
concentration. The two linear parts of the saturation curve intercept
at a ratio enzyme:substrate 1.1. Dashed line,
fully reduced enzyme obtained from anaerobic reaction with 50 µM cholesterol. Bottom panel:
, oxidized
(8Cl-FAD)-H69A BCO in 100 mM potassium phosphate buffer, pH
7.5, at 15 °C; long dashed line, semiquinone;
and dashed line, fully reduced forms obtained by
photoirradiation as described for wild-type BCO.
Spectral and redox properties of wild-type, H69A, and
(8Cl-FAD)-H69A forms of BCO
176 mV) and phenosafranine (Em =
239 mV).
11 (7), a value higher than that
(pKa
10) of free FAD (21). For H69A BCO a similar
value was determined (pKa
11.1 ± 0.1). This
confirms the absence of an effect of the covalent linkage specifically
on this ionization process. The increase in pK observed for
wild-type and H69A BCO compared with free FAD is likely to result from
the microenvironment around the oxidized flavin pyrimidine: N(3)-H
forms a tight H-bond (2.8 Å) with the backbone His202
C(1)=O. The Arg477 guanidinium group is at 3.1 Å from the
flavin C(4)=O and can make hydrogen bond contacts directly with the
side chains of both Glu475 and Glu311, this
interaction likely neutralizing its charge.2
30% residual activity observed with wild-type BCO at 20%
propan-2-ol after 300 min, where H69A BCO is completely
inactivated.
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Fig. 2.
Stability of wild-type and H69A BCO and
effect of propan-2-ol. The enzymes, 0.1 mg protein/ml, in 50 mM potassium phosphate buffer, pH 7.5, and at 25 °C were
incubated at the following concentrations of propan-2-ol: 0% ( );
10% (
); 20% (
); 30% (
); and 40% (
) (all final
concentrations).
5-6
4-5 isomerization step is not
rate-limiting for either enzyme, the kcat for
the isomerization step being 150 s
1 and 28 s
1 for wild-type and H69A BCO, respectively.
Kinetic parameters for the reaction of wild-type, H69A, and
(8Cl-FAD)-H69A forms of BCO with cholesterol (oxidation) and
5-cholesten-3-one (isomerization)
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Scheme 1.
Reactions catalyzed by BCO. In the
first step (left-hand side) cholesterol is dehydrogenated to
the intermediate 5-cholesten-3-one; the redox equivalents are used to
reduce the oxidized flavin, and are subsequently transferred onto
oxygen (oxidase reaction). Subsequently a ( 5-6
4-5)
isomerization of the intermediate to form 4-cholesten-3-one occurs
(right-hand side) at the level of oxidized enzyme flavin.
Note that with BCO the isomerization reaction and reoxidation of
reduced enzyme by oxygen are substantially faster than the
dehydrogenation, the latter being rate-limiting.
15% of the total change (data not shown). The value is
significantly smaller than the one observed for the wild-type BCO
(
60%) which is due to the particular kinetic situation in which the
rates of flavin reduction and reoxidation are similar (10). The smaller
amplitude of the 446 nm absorbance change observed with the H69A BCO
indicates that in the noncovalent mutant the rate of enzyme reduction
is significantly decreased in comparison to the wild-type enzyme. The
initial decrease in absorption was followed by a stationary phase, the
duration of which depends on the initial cholesterol concentration and
which leads to the fully reduced enzyme as the final state. The 446-nm
traces were analyzed as a function of oxygen concentration according to
Ref. 15; the kinetic parameters obtained are given in Table
III. The double-reciprocal
(Lineweaver-Burk) plot of the turnover number as a function of
substrate concentration always gave a set of parallel lines, suggesting
that a ping-pong mechanism is still operative for the H69A BCO. EMTN
experiments confirm that the absence of covalent flavin linkage in BCO
results in a large decrease in the turnover number and in a large
increase in the
Chol term of the steady state equation
(Table III).
Comparison of steady-state coefficients obtained for the reaction of
wild-type and H69A BCO with cholesterol as substrate in
stopped-flow experiments
8
M was estimated, indicating a rather tight binding. It is
worth noting that the data are consistent with a 1:1 molar ratio of apoprotein:FAD binding. The kinetics of reconstitution of the H69A
apoprotein-FAD complex were studied under pseudo-first order conditions
(10-150-fold excess of FAD or apoprotein) by following the time course
of quenching of protein or flavin fluorescence. In both cases the
reaction curve is biphasic and is best fit by two exponentials. Fig.
3 shows the time course of the total
change in protein fluorescence after mixing H69A apoprotein with excess of FAD: both the initial fast phase and the slow secondary change proceed according to a first-order exponential processes.
Interestingly, at the end of the reaction, FAD fluorescence was
quenched to 10-15% of the value of free FAD (see above). The observed
first-order rate constant of the fast phase
(kobs1) depends linearly on the concentration of
the FAD (inset of Fig. 3). The process can thus be described
by two subsequent, irreversible steps with constants k1 = 1.5 × 104
M
1 s
1, and
k2 = 2 × 10
4
s
1. Although in this context the reversal rate constants
can be assumed to be close to zero, they have a finite value since the holoenzyme reconstitution process is reversible. The observed first-order rate constant for the second (slow) phase was not dependent
on the apoprotein concentration and represents a sequential rearrangement to yield the holoenzyme form. The protein fluorescence change associated with the second phase corresponds to
25% of the
total quenching during the reconstitution of H69A apoprotein with FAD.
Very similar results were obtained by monitoring changes in protein
fluorescence after mixing apoprotein with a large excess of FAD
(10-150-fold).
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Fig. 3.
Time course of fluorescence quenching during
the reconstitution of H69A BCO apoprotein with FAD. Protein
fluorescence change (arbitrary units) after the mixing of 1.25 µM apoprotein solution with 12.6 µM FAD
solution (final concentrations) in 100 mM potassium
phosphate buffer, pH 7.5, at 15 °C. Excitation wavelength: 285 nm;
emission wavelength: 340 nm. Inset, effect of FAD
concentration on the observed first-order rate constant
(kobs1) of the reconstitution of 0.63 µM H69A BCO apoprotein. The values of
kobs1 were calculated from the slope of the
first rapid phase of the logarithmic plots as shown in the main
graph, after subtraction of the second, slow phase.
115 mM that compares to 0.14 mM for wild-type BCO.
These properties are comparable to those observed with CO from S. hygroscopicus that contains noncovalently linked FAD (7).
15 mV (22) and, thus, the redox potentials
for each single electron transfer cannot be determined accurately. The
midpoint potential of the oxidized/reduced forms (Em = (E1 + E2)/2) was
determined by using phenosafranine and cresyl violet as reference dyes.
The redox potential difference with respect to the dye was calculated by plotting log([dyeox]/[dyered]) as a
function of
log([Eox]/[Ered]) (18): this plot has a slope
0.88 (data not shown) which is close to
the theoretical value of 1.0 for a two-electron reduction process. H69A
BCO has a midpoint redox potential, Em, which is
about 100 mV more negative than wild-type BCO (Table I). This change
should influence catalysis, since the midpoint potential of
200 mV
makes enzyme reduction by cholesterol, the rate-limiting step with
wild-type BCO (10), thermodynamically unfavorable. A decrease in the
reoxidation rate is not an issue because the
O2/H2O2 couple is more positive
(+300 mV at pH 7) than the FAD/FADH2 couple. The results
indicate that the lack of the flavin covalent link results in a
100
mV shift of the midpoint redox potential (
101 mV versus
204 mV for the wild-type and H69A, respectively). A similar change in
redox potentials has been recently reported for vanillyl-alcohol
oxidase (23).
View larger version (19K):
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Fig. 4.
Time course of anaerobic reduction of H69A
BCO using the xanthine/xanthine oxidase system. Selected spectra
obtained during the course of the anaerobic reduction of 8.7 µM H69A BCO in 100 mM potassium phosphate
buffer, pH 7.5, in the presence of 200 µM xanthine and 5 µM BV, at 15 °C: solid line, before
addition xanthine oxidase; dotted line, 12.5 min, dot
dash line, 22.5 min; long dashed line, 42.5 min, and
dashed line, 62.5 min after addition of 10 nM
xanthine oxidase.
152 mV
versus
207 mV). Binding of the analogue was followed by
the quenching of the protein fluorescence and is similar
(Kd = 1.6 × 10
8 M)
to that of normal FAD. The spectra of the oxidized and
cholesterol-reduced (8Cl-FAD)-H69A BCO are intermediate in shape
compared with those of wild-type and H69A BCO (Fig. 1). This suggests
that the peculiar spectral properties of H69A BCO result from a
combination of electronic and structural effects. The spectrum of the
radical intermediate, on the other hand, is similar to that of
wild-type BCO (Fig. 1), suggesting that electronic factors are decisive
for the properties of the semiquinone. With (8Cl-FAD)-H69A BCO, a
higher amount of flavin semiquinone (~40%) is observed than is
formed with H69A BCO. Sulfite reactivity strongly differentiates
(8Cl-FAD)-H69A BCO from that containing normal FAD. The former reacts
rapidly to form the flavin N(5)-covalent adduct with a
Kd
3.6 mM that is intermediate
between the binding constants for wild-type and H69A BCO containing
unmodified FAD. Catalytic activity of the (8Cl-FAD)-H69A BCO is
3.5-fold higher than that observed with normal FAD reconstituted
mutant, while the rate of isomerization is unaltered by the flavin
substitution (Table II).
160 mV and was
determined using the xanthine/xanthine oxidase reducing system and
cresyl violet as mediator. This potential is thus
45 mV more positive than that of H69A containing normal FAD. Hence, the higher catalytic activity of the (8Cl-FAD)-H69A BCO is due largely to its more
positive redox potential.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 × 10
8 M for both FAD- and
(8Cl-FAD)-apoprotein complexes was determined. The lack of the covalent
flavinylation due to the substitution of H69A does not abolish the
tight binding of the coenzyme to BCO. This observation demonstrates
that a functional holoenzyme can be obtained from the noncovalent
interactions of the coenzyme with the apoprotein moiety. Binding of FAD
to H69A apoprotein appears to be a two-stage process, in which only the
first (rapid) phase depends on the FAD or apoprotein concentration, a
reconstitution process similar to that of many other noncovalent
flavoproteins (24, 25). The rapid initial phase observed in the
fluorimetric experiments leads directly to the fully functional enzyme
since it has the same rate as the recovery of CO activity. The second step might reflect secondary conformational changes of the holoenzyme (24).
30-fold decrease in the rate of flavin reduction. By
comparison of the parameters of cholesterol dehydrogenation with those
for the isomerization of 5-cholesten-3-one (Table II, see also Scheme
1), it is apparent that the latter is never rate-limiting.
204 mV)
is very close to that of CO from S. hygroscopicus (
217 mV)
that contains noncovalent FAD (7) and to that of free FAD (
207 mV).
The effect of the 8
-(N3)histidyl substitution on the redox potential
of free flavins consists of an increase of
30 mV (26). It should be
noted, however, that this effect is strongly pH dependent and reflects
the ionization state of the histidine substituent (27). These authors
have pointed out that a role of histidine substitution might indeed be
the modulation of the redox potential of the flavin-bound enzyme. This
suggestion appears to be borne out by the present results. On the other
hand the rates of flavin reduction for BCO and SCO (235 s
1 and 232 s
1 at 1% Thesit and 10%
propan-2-ol, respectively) (10) do not correlate with the corresponding
midpoint redox potentials. This apparent inconsistency might be due to
the mentioned potentials having been determined for the free enzyme
forms as opposed to the enzyme-substrate systems. Furthermore, it is to
be expected that additional factors such as the nature and position of
functional groups involved in catalysis and parameters derived from the
overall structures are important in determining the absolute values of catalytic steps.
4-fold higher catalytic activity than
the corresponding FAD derivative (Table II) which probably stems from
the
45 mV more positive redox potential (Table I). Thus, within the
same enzyme system a correlation of rate of reduction with the redox potential appears to be verified. On the other hand, the rate of
5-6
4-5 isomerization that does not involve redox processes is unaffected by the coenzyme replacement (Table II).
-substitution with a
(N3)histidine in BCO is an important factor in the modulation of its
redox properties as a way to increase its oxidation power. Why there
are two CO enzymes in the genus Brevibacterium that are
differentiated by the presence of the covalent flavin linkage is not
known. Whether this is a fortuitous event or reflects evolutive pressure (i.e. due to the different environments in which
the two COs reveal their catalytic function) is an interesting aspect awaiting elucidation.
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FOOTNOTES |
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* This work was supported by grants from the Italian MURST (to L. P. and M. S. P.).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: Dept. of Structural and Functional Biology, University of Insubria, via J.H. Dunant 3, 21100 Varese, Italy. Tel.: 39-0332-421506; Fax: 39-0332-421500; E-mail: loredano.pollegioni@uninsubria.it.
Published, JBC Papers in Press, February 28, 2001, DOPI 10.1075/jbc.M010953200
2 R. Coulombe, K. Q. Yue, S. Ghisla, and A. Vrielink, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are:
CO, cholesterol
oxidase;
SCO, cholesterol oxidase from S. hygroscopicus;
BCO, recombinant cholesterol oxidase from B. sterolicum
expressed in E. coli;
cholesterol, 5-cholestene-3-ol;
EMTN, enzyme monitored turnover;
EFlox, oxidized enzyme;
EFlseq, enzyme flavin
semiquinone;
EFlred, reduced enzyme;
8Cl-FAD, 8-chloro-FAD.
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REFERENCES |
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