From the Departments of Biochemistry and
Chemistry, University of Leicester, University Road,
Leicester LE1 7RH, the § Department of Pure and Applied
Chemistry, University of Strathclyde, The Royal College, 204 George
Street, Glasgow G1 1XL, and the ** Department of Chemistry, University
of Edinburgh, The King's Buildings, West Mains Road,
Edinburgh EH9 3JJ, United Kingdom
Received for publication, December 1, 2000, and in revised form, March 23, 2001
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ABSTRACT |
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The midpoint reduction potentials of the FAD
cofactor in wild-type Methylophilus methylotrophus (sp.
W3A1) electron-transferring flavoprotein (ETF) and the Electron-transferring flavoproteins
(ETFs)1 act as carriers of
electrons in bacteria and mitochondria. They mediate electron transfer between degradative enzymes and membrane-bound electron acceptors (1). ETFs have been classified into two functional groups
(2). Housekeeping ETFs function in the oxidation of fatty acids and
some amino acids and have been isolated from mammalian and bacterial
sources (1, 3). Specialized ETFs are restricted to prokaryotes and are
synthesized under defined nutritional conditions. Specialized
ETFs are involved in the oxidation of trimethylamine (4) and carnitine
(5, 6) and are also important in nitrogen fixation (7). All ETFs
possess one equivalent of non-covalently bound FAD per ETF heterodimer,
except the ETF from Megasphaera elsdenii, which contains 2 equivalents of FAD per dimer (8). It has been shown that AMP (1 equivalent) is associated with the housekeeping ETFs from pigs (9),
humans (10), and Paracoccus denitrificans (11) and
with the specialized ETF from Methylophilus methylotrophus
(12).
Mammalian and bacterial ETF proteins act as one-electron
carriers, cycling between the oxidized and anionic flavin semiquinone forms. The ETF from M. elsdenii is unusual in acting
physiologically as a two-electron carrier. ETF from mammalian sources
and P. denitrificans can be reduced to the dihydroquinone
form, by reduction with dithionite or by photoreduction (13-15),
although reduction to the two-electron level is relatively slow.
M. methylotrophus ETF is readily converted to the
semiquinone form in reactions with its physiological electron donor,
trimethylamine dehydrogenase (TMADH) (4), or during artificial
reduction with dithionite (16). However, further reduction to the
dihydroquinone is not observed with dithionite (16) or with catalytic
amounts of TMADH (4). Reduction of M. methylotrophus ETF to
the dihydroquinone form can be achieved (albeit sluggishly) by
electrochemical methods (17). In addition, when ETF is in complex with
TMADH, the FAD is more readily reduced to the two-electron level (18).
In this latter case, further reduction to the dihydroquinone is likely
to be a consequence of a large scale structural reorganization in
ETF that accompanies complex assembly with TMADH (18-20).
The midpoint reduction potentials of the E'1
(quinone-semiquinone) and E'2
(semiquinone-dihydroquinone) couples of the FAD in M. methylotrophus ETF have been determined. The potential of the
quinone-semiquinone couple is exceptionally high (+196 mV (17) and +141
mV (21) as determined by electrochemical and spectrophotometric
methods, respectively), consistent with a need to accept electrons from
the 4Fe-4S center of TMADH (midpoint potential, +102 mV (22)). The
potential of the semiquinone-dihydroquinone couple is more conventional
(-197 mV; (17)), indicating that there is a substantial (and
essentially complete) kinetic block on full reduction of ETF by
dithionite (-530 mV) or photoexcited deazariboflavin (-650 mV). A
similar, albeit less complete, kinetic block on reduction to the
dihydroquinone has been reported for pig liver ETF; reduction to the
dihydroquinone level in pig liver ETF requires about 1 h for
equilibration (13, 14). The potentials measured for free M. methylotrophus ETF (as with any ETF) may not of course reflect the
situation in the electron transfer complex with its physiological
electron donor (TMADH) but nevertheless are likely to serve as a
reasonable guide.
M. methylotrophus ETF shares considerable sequence identity
with bacterial and mammalian ETFs (10, 11). Preliminary
crystallographic studies of M. methylotrophus ETF have been
reported (23), but to date no crystallographic structure for the
protein is available. However, crystallographic structures of human and
P. denitrificans ETF have been determined at 2.1 Å (10) and
2.6 Å (11) resolution, respectively. Using the x-ray structure of
human ETF as a template (10), we built a model of the structure of
M. methylotrophus ETF, in free solution and in complex with
TMADH (19). The model predicts that the two subunits (subunit Isolation of the Potentiometry--
Redox titrations were performed within a
Belle Technology glove box under a nitrogen atmosphere (oxygen
maintained at <5 ppm) in 50 mM potassium phosphate buffer,
pH 7.2. Anaerobic titration buffer was prepared by flushing freshly
prepared buffer with oxygen-free nitrogen. Protein samples admitted to
the glove box were deoxygenated by passing through a Bio-Rad 10DG
column, with final dilution of the eluted protein to give an ETF
concentration of 70-80 µM. Solutions of benzyl viologen,
methyl viologen, 2-hydroxy-1,4-naphthaquinone, and phenazine
methosulfate were added to a final concentration of 0.5 µM as redox mediators for the titrations. Absorption
spectra (300-750 nm) were recorded on a Shimadzu 2101 UV-visible
spectrophotometer, and the electrochemical potential was monitored
using a CD 740 m combination pH/voltmeter coupled to a Russell
platinum/calomel electrode. The electrode was calibrated using
the Fe(II)/Fe(III)-EDTA couple (108 mV) as a standard. The flavoprotein
solutions were titrated electrochemically using sodium dithionite as
reductant and potassium ferricyanide as oxidant, as described by Dutton (24). After the addition of each aliquot of reductant, and allowing equilibration to occur (stabilization of the observed potential), the
spectrum was recorded, and the potential was noted. The process was
repeated at several (typically ~40) different potentials. In this
way, a set of spectra representing reductive and oxidative titrations
was obtained. Small corrections were made for any drift in the base
line by correcting the absorbance at 750 nm to zero, and
spectrophotometric contributions from the mediators were removed prior
to data analysis. The observed potentials were corrected to those for
the standard hydrogen electrode (platinum/calomel + 244 mV).
Treatment of Data--
Data manipulation and analysis were
performed using Origin software (Microcal). Absorbance values at
wavelengths of 470 nm (near the oxidized flavin maximum) and 370 nm
(close to the maximum for the anionic semiquinone) were plotted against
potential. Data were fitted to Equation 1, which represents a
two-electron redox process derived by extension to the Nernst equation
and the Beer-Lambert law, as described previously (24, 25).
Purification of the
Consistent with previous reports, anaerobic titration of wild-type ETF
with sodium dithionite (16) or enzymatic reduction with TMADH (4)
reduces the protein only to the level of the flavin anionic semiquinone
(Fig. 3A). The addition of
excess dithionite (6× molar excess) does not reduce the protein
further to the dihydroquinone form, even following prolonged incubation
(30 min). The potential of the E'2 couple for
wild-type ETF is almost certainly more negative than Potentiometric Titrations of Wild-type ETF--
In all cases,
titrations were initiated from fully oxidized ETF and proceeded
gradually to the end point of the titration by the addition of small
aliquots of sodium dithionite (from 1 and 10 mM stocks) and
then back again to oxidized ETF by addition of aliquots of potassium
ferricyanide stocks of the same concentration. The protein samples
remained completely soluble and stable throughout the course of the
titration, enabling collection of good quality sets of spectra. No
hysteretical effects were observed in any of the redox titrations.
Spectra recorded at similar potentials during oxidative and reductive
titrations were essentially identical. Representative spectra for the
reductive titration of wild-type ETF and plots of the absorbance
versus potential are shown in Fig.
4, A and B.
In the reductive titration with wild-type ETF in the presence of
mediators, reduction occurs first to the flavin anionic semiquinone, and a small proportion of the flavin is subsequently reduced to the
dihydroquinone (Fig. 4A). Partial reduction to the
dihydroquinone does not occur in the absence of mediators (Fig. 3),
with reduction taking place only to the level of the flavin anionic
semiquinone; this requires further comment. The time taken to complete
reductive titrations in the potentiometry experiments was typically
around 3-4 h. This is clearly much longer than the time taken to
complete the simple spectral analysis shown in Fig. 3. Redox mediators were also included in the potentiometric analysis but were absent in
the spectroscopic characterization shown in Fig. 3. The presence of
redox mediators and also the long time period for protein reduction ensured more complete equilibration of the system in the potentiometric analyses, but this is clearly not the case with the spectral changes displayed in Fig. 3. Notwithstanding the inclusion of redox mediators and the prolonged time periods of the potentiometry measurements, the
spectral changes accompanying reduction of wild-type ETF in the
presence of redox mediators indicates that complete
reduction to the dihydroflavin is not obtained. This is indicated by
the presence of considerable semiquinone signature at 370 nm at the end
of the titration. Further addition of dithionite did not reduce the
absorption at 370 nm to the level seen for the
Titrations performed in the presence and absence of mediators serve to
illustrate the kinetic limitation on reduction to the dihydroquinone
form in wild-type ETF, a kinetic bottleneck that can be overcome (at
least in part) by the inclusion of redox mediators during the course of
reductive titration. That equilibration was achieved with wild-type ETF
in the majority of the potentiometric analyses is evident from the
stability of the potential readings throughout the reductive titration
and from the well defined transitions (Fig. 4B). The lack of
hysteresis on performing the oxidative titration likewise indicates
that equilibration was achieved (except at very low potentials) and
also that FAD was not released from ETF during the course of the
potentiometric titrations. The midpoint reduction potentials for
E'1 and E'2 were obtained
by fitting the data shown in Fig. 4A to Equation 1. These
potentials are compared with values obtained by other workers for
wild-type M. methylotrophus and mammalian ETFs in Table
I.
Potentiometric Titrations of
The spectral changes observed during reductive titration (over 3-4 h)
are again different from those observed in the spectroscopic characterization of the
During reductive titration of the
We are confident that equilibration is achieved in the potentiometric
studies of the Reduction of Developing a better understanding of the control of reduction
potential of redox centers in proteins is pivotal to our appreciation of the mechanisms of biological electron transfer. Not surprisingly, the simple electron transfer proteins (e.g. the rubredoxins
and flavodoxins) have proved to be tractable model systems for studies of this type. The control of reduction potential for large centers such
as flavin is particularly challenging, given the number of potential
interactions it can make with the protein. The large number of
potential contacts between the protein and flavin probably accounts for
the extensive range of potentials observed in flavoproteins. The
structural simplicity of the flavodoxin family of flavoproteins has
made them attractive models for establishing the relationship between
oxidation-reduction properties of the flavin and its interactions with
the apoprotein. The flavodoxins shuttle between the semiquinone and
hydroquinone states, and they exhibit the lowest reduction potentials
among the flavoprotein family, with values recorded as low as In this paper we have initiated a study of the control of redox
potential in another simple flavoprotein, ETF. ETF is an attractive model system because of the exceptionally high potential of the oxidized-semiquinone couple (cf. the flavodoxins, where this
couple is exceptionally low in potential) and the known large scale
conformational dynamics of ETF, which probably affect the redox
properties of this protein. The midpoint reduction potential of the
E'1 (oxidized-semiquinone) couple of M. methylotrophus (sp. W3A1) ETF is the most
positive couple for any known flavoprotein. Identification of the
environmental effects around the flavin isoalloxazine ring that
contribute to this extreme stabilization of the flavin anionic
semiquinone is of major interest in terms of understanding the
mechanism and from the viewpoint of engineering novel flavoenzymes with
altered redox properties. Human ETF, which is highly related to
M. methylotrophus ETF, does not stabilize the flavin
semiquinone to anywhere near the same extent (Table I). In P. denitrificans ETF, the E'1
(oxidized-semiquinone) couple is not widely separated from the
E'2 (semiquinone-dihydroquinone) couple, and the
value of the two-electron oxidation-reduction potential has been
determined as The role of flavin contact residues in human ETF in
determining the values of the E'1 and
E'2 couples has been investigated (37).
The potentiometric and other reductive titrations we have performed
highlight the importance of structural movement in the ETF protein,
because kinetic as well as thermodynamic factors affect the reduction
of the flavin. These data are entirely consistent with our x-ray
solution scattering studies of wild-type ETF that indicate that the
molecule is highly dynamic (20) and that it populates an ensemble of
conformations in which domain II (the flavin-binding domain) rotates
around two hinge regions at its interface with domains I and III. By
breaking the predicted (inferred from our structural model of M. methylotrophus ETF) salt link between An unusual feature of the flavin environment in human and P. denitrificans ETF is the presence of an intraflavin hydrogen bond
between the ribityl 4' hydroxyl and the flavin N1 (10, 11). The role of
this novel hydrogen bond in stabilizing the potentials of the flavin
has been probed by exchanging FAD for 4'-deoxy-FAD in human ETF (38).
By incorporating 4'-deoxy-FAD in human ETF a destabilization of the
oxidized-semiquinone couple by 0.116 V is observed, indicating that the
novel hydrogen bond stabilizes the flavin semiquinone. In this altered
form of human ETF, reduction to the dihydroquinone is slow and
incomplete, suggesting a kinetic block on full reduction, analogous to
that seen with M. methylotrophus ETF, and to a lesser
extent, pig liver ETF (13, 14). The kinetic limitation on reduction to
the dihydroquinone probably reflects conformational differences in
protein structure that occur during reduction of the FAD. Whether
M. methylotrophus ETF contains the unusual intraflavin
hydrogen bond is uncertain and must await a crystallographic
determination of the structure of the protein, but the potential
absence of such a bond may account (at least in part) for the
kinetic limitation on achieving full reduction in wild-type ETF. The
interplay between residue Concluding Remarks--
Mutagenesis of R237A mutant
were determined by anaerobic redox titration. The FAD reduction
potential of the oxidized-semiquinone couple in wild-type ETF
(E'1) is +153 ± 2 mV, indicating
exceptional stabilization of the flavin anionic semiquinone species.
Conversion to the dihydroquinone is incomplete
(E'2 <
250 mV), because of the presence of
both kinetic and thermodynamic blocks on full reduction of the FAD. A
structural model of ETF (Chohan, K. K., Scrutton, N. S., and
Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231-236) suggests that the guanidinium group of Arg-237, which is
located over the si face of the flavin isoalloxazine ring, plays a key role in the exceptional stabilization of the anionic semiquinone in wild-type ETF. The major effect of exchanging
Arg-237 for Ala in M. methylotrophus ETF is to engineer a
remarkable ~200-mV destabilization of the flavin anionic semiquinone
(E'2 =
31 ± 2 mV, and
E'1 =
43 ± 2 mV). In addition,
reduction to the FAD dihydroquinone in
R237A ETF is relatively
facile, indicating that the kinetic block seen in wild-type ETF is
substantially removed in the
R237A ETF. Thus, kinetic (as well as
thermodynamic) considerations are important in populating the redox
forms of the protein-bound flavin. Additionally, we show that
electron transfer from trimethylamine dehydrogenase to
R237A ETF is severely compromised, because of impaired assembly of
the electron transfer complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(residues 1-321) and subunit
(residues 322-585)) of M. methylotrophus ETF comprise three domains. Domain I (the
N-terminal region of the
subunit), domain II (the C terminus of the
subunit and a small C-terminal region of the
subunit), and
domain III (the majority of the
subunit) form a Y-shaped structure,
with domains I and III forming a shallow "bowl" in which domain II
rests. Domain II is connected to domains I and III by two flexible
regions of polypeptide chain (19). Small angle x-ray scattering studies
have demonstrated that domain II is mobile with respect to domains I
and III (20). The isoalloxazine ring of FAD interacts almost
exclusively with domain II (Fig. 1). The
model of M. methylotrophus ETF suggests that residue Arg-237
is located close to the FAD isoalloxazine ring, with its guanidinium
group positioned over the si face of the dimethylbenzene
subnucleus. The guanidinium group is thus located to help stabilize the
increased electron density (which resides predominantly in the
pyrimidine subnucleus) on reduction of the flavin to the anionic
semiquinone. In this paper, we report the redox properties of a mutant
ETF in which Arg-237 is replaced by Ala. We show that Arg-237 plays a
key role in the exceptional stabilization of the anionic semiquinone in
native ETF and that mutation of Arg-237 to Ala removes the kinetic
block to full reduction of the FAD. These findings demonstrate that the
chemical properties of a single residue close to the flavin
isoalloxazine ring can have profound effects on the redox properties
(thermodynamic and kinetic) of protein-bound flavin.
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Fig. 1.
Amino acid differences around the FAD
isoalloxazine ring binding site in (A) human and
(B) M. methylotrophus ETF. Note
(i) the potential intersubunit salt bridge ( Arg-237-
Glu-37) and
(ii) the presence of an additional positive charge (
Lys-247) in
M. methylotrophus ETF.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R237A Mutant and Expression of M. methylotrophus ETFs in Escherichia coli--
Isolation of the
R237A
mutant form of ETF was performed using the QuikChange site-directed
mutagenesis kit supplied by Stratagene and oligonucleotides 5'-CTT TGC
TGC TCA GCT CCG ATT GCG GAT-3' and 5'-ATC CGC AAT CGG AGC TGA GCA GCA
AAG-3'. The ETF expression plasmid pED1 (20) was used as template DNA
for the mutagenesis reaction. Recombinant wild-type ETF was expressed
from plasmid pED1 in E. coli strain TG1 as described (20).
To ensure that no spurious changes had arisen as a result of the
mutagenesis reaction, the entire ETF gene was resequenced using the
Amersham Pharmacia Biotech T7 sequencing kit and protocols.
Recombinant wild-type and mutant ETF proteins were expressed from
plasmids pED1 and pED1R237A, respectively, in the E. coli
strain TG1. Recombinant strains were grown at 20 °C in 2xYT
medium supplemented with 100 µg/ml ampicillin. ETF was
purified in large quantities (~30 mg/liter of late exponential phase
culture) from recombinant strains of E. coli. Harvested
cells were resuspended in buffer A (50 mM potassium phosphate buffer, pH 7.2, 0.2 mM EDTA) and broken in a
French press (140 megapascals, 4 °C). The extract was clarified by
centrifugation at 15,000 × g for 90 min, and solid
ammonium sulfate was added to 50% saturation. The precipitate was
removed by centrifugation, and the supernatant was applied to a high
performance phenyl-Sepharose column using a fast protein liquid
chromatography system (Amersham Pharmacia Biotech) previously
equilibrated with buffer A containing 1.5 M ammonium
sulfate. After being washed with equilibration buffer, protein
was eluted using a descending gradient (1.5 to 0 M) of
ammonium sulfate. Fractions containing ETF were dialyzed exhaustively
against buffer A and applied to a Q-Sepharose column equilibrated with
buffer A. After washing with buffer A, protein was eluted using a
gradient (0 to 2 M KCl); ETF was eluted at ~0.5
M KCl. Samples were dialyzed exhaustively against potassium phosphate buffer, pH 7.2 and stored (-70 °C) in the presence of 20%
ethylene glycol.
In Equation 1, A is the total absorbance;
a, b, and c are component absorbance
values contributed by one flavin in the oxidized, semiquinone, and
reduced states, respectively. E is the observed potential;
E'1 and E'2 are the
midpoint potentials for oxidized-semiquinone and semiquinone-reduced
couples, respectively. In using Equation 1 to fit the
absorbance-potential data, the variables were unconstrained, and
regression analysis provided values in close agreement with those of
the initial estimates.
(Eq. 1)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R237A Mutant ETF and Reduction of Wild-type
and
R237A ETF with Dithionite--
The
R237A mutant ETF was
purified essentially as described for the wild-type protein. A notable
difference between the mutant and wild-type proteins is the redox state
of the flavin.
R237A ETF is purified in the oxidized form, whereas
wild-type ETF is isolated as a mixture of oxidized and anionic flavin
semiquinone forms (Fig. 2). Oxidation of
the wild-type ETF with potassium ferricyanide, followed by immediate
rapid gel filtration to remove the oxidant, generates the oxidized form
of wild-type ETF. A comparison of the spectral properties of the
oxidized wild-type and
R237A mutant ETF proteins indicates that the
peak of flavin absorption (446 nm) in the
R237A mutant is shifted
compared with the corresponding peak (438 nm) in wild-type ETF. In
addition, the A388/A446
ratio (1.01) for
R237A ETF is greater than the
A380/A438 ratio (0.89) for wild-type ETF. These observations suggest that the isoalloxazine ring of FAD in
R237A ETF is more exposed to solvent than in
wild-type ETF (26), an observation that is consistent with the
structural model for M. methylotrophus ETF (19).
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Fig. 2.
Spectra of oxidized M. methylotrophus ETF and the as-purified
R237A mutant ETF. Conditions: 50 mM potassium phosphate buffer, pH 7.0. wt, wild
type.
250 mV
(see below) but is more positive than the reduction potential of
dithionite (-530 mV). This indicates that there is a substantial
kinetic block on full reduction of the flavin. By contrast, reduction
of the
R237A mutant ETF with dithionite proceeds to full reduction
(Fig. 3, B and C). The two reductive phases (oxidized-semiquinone and semiquinone-dihydroquinone couples) are
clearly resolved. The spectral changes accompanying reduction of the
oxidized FAD to the anionic semiquinone have isosbestic points at 491 and 391 nm, and a single isosbestic point at 342 nm is seen for
reduction of the anionic semiquinone to the dihydroquinone.
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Fig. 3.
Spectral changes accompanying reduction of
wild-type and R237A ETF proteins with sodium
dithionite. A, spectral changes for wild-type ETF on
titrating with dithionite. Inset, plot of absorbance
change at 438 nm (filled circles) and 370 nm (filled
triangles) versus mole ratio of dithionite added to
flavin content. B, spectral changes for conversion of
oxidized
R237A ETF to the anionic semiquinone form. C,
spectral changes for conversion of anionic semiquinone
R237A to the
dihydroquinone form. Conditions: 50 mM potassium phosphate
buffer, pH 7.0.
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Fig. 4.
Representative spectra for the reduction of
wild-type and R237A ETF proteins during the
course of potentiometric titrations and plots generated from
potentiometric titration. A, spectral changes accompanying
titration of wild-type ETF during potentiometric measurements (for
clarity, only selected recorded spectra are shown). C,
spectral changes accompanying titration of
R237A ETF during
potentiometric measurements (for clarity, only selected recorded
spectra are shown). B, plot of absorbance change at 470 nm
(open circles) and 370 nm (filled circles) for
wild-type ETF. D shows a similar plot for data from the
R237A ETF redox titration. These data were fitted using Equation 1.
Values of midpoint reduction potentials are given in Table I.
Conditions: 50 mM potassium phosphate buffer, pH 7.0, 20 °C. Abs., absorbance
R237A mutant. That a
substantial proportion of the wild-type ETF is reduced beyond the
anionic semiquinone level, however, is indicated by the extent of
bleaching in the absorption range of 440-470 nm (compared with
titrations in the absence of mediators) and the partial bleaching of
the absorption at 370 nm (Fig. 4, A and B). The
inability to completely reduce wild-type ETF probably reflects the
presence of a substantial kinetic block on full reduction for a
proportion of the protein sample.
Measured midpoint reduction potentials for wild-type and R237A M. methylotrophus (sp. W3A1) ETF and comparison with the
midpoint reduction potentials of ETF proteins from other species
R237A ETF--
As with wild-type
ETF,
R237A ETF remained soluble throughout the course of reductive
and oxidative titrations, and no hysteresis was observed during the
course of reduction by dithionite and reoxidation by ferricyanide.
Representative spectra during the course of reductive titration
with dithionite are shown in Fig. 4B, and plots of
absorbance versus potential are shown in Fig. 4D.
R237A ETF shown in Fig. 3, B and
C. In Fig. 3, the anionic semiquinone species is populated
prior to full reduction to the dihydroquinone, whereas in
potentiometric titrations full development of the anionic
semiquinone signature at 370 nm is not observed (Fig. 4,
C and D). Again, we attribute this to a kinetic
limitation that prevents rapid reduction to the dihydroquinone in the
absence of mediators (see also the value for E'2
below). However, mutation of
Arg-237 to Ala partially relieves the
kinetic block, because the dihydroquinone clearly does form in the
R237A mutant enzyme during the reductive titration performed without
mediators (Fig. 3C), unlike wild-type ETF, which is reduced
only to the level of the flavin semiquinone (Fig. 3A). We
conclude, therefore, that
Arg-237 contributes to the kinetic block
on reduction to the dihydroquinone seen in wild-type ETF.
R237A mutant enzyme, small amounts
of red anionic semiquinone are observed, as evidenced by very small
increases in absorption at 370 nm during the early phase of reduction
(Fig. 4D). The data at this wavelength fit to a two-electron
Nernst function, and the two one-electron reduction steps are resolved
as semiquinone (E'1 (oxidized-semiquinone) =
43 ± 2 mV, with E'2
(semiquinone-dihydroquinone) =
31 ± 2 mV). Clearly, there
is a large (200 mV) perturbation of the reduction potential of the
R237A ETF compared with wild-type. Thus, the mutation appears to
relieve both thermodynamic and kinetic blocks to full reduction of the flavin.
R237A mutant ETF, as reflected in the stability of
the potential readings, the well defined absorbance transitions (Fig.
4D), and the lack of hysteresis in reductive and oxidative
titrations. Relative values for the redox couples in wild-type and
mutant enzymes are presented in Table I.
R237A by TMADH--
Reduction of
R237A and
wild-type ETF by catalytic amounts of TMADH was followed under
anaerobic conditions (Fig. 5). Complete reduction of wild-type ETF to the anionic semiquinone form was achieved
in less than 5 min; further reduction to the dihydroquinone was not
observed. Using the same experimental conditions, reduction of the
R237A mutant ETF required 24 h to effect the almost complete reduction of the protein. The spectral changes accompanying reduction of
R237A ETF by TMADH are similar to those observed in the
potentiometric studies of this protein (Fig. 4C). These data
reinforce the assertion that the accumulation of the anionic
semiquinone species for the
R237A ETF observed in dithionite
titrations of the protein (Fig. 3B) is attributed to
non-equilibration of electrons. A priori, the very sluggish
rate of reduction of
R237A ETF may be expected from the unfavorable
driving force for the electron transfer reaction. This driving force
can be calculated from the midpoint reduction potential of the 4Fe-4S
center of TMADH (+102 mV; (22)) and the E'1 and
E'2 couples of
R237A ETF (Table I). The
driving forces for electron transfer from the 4Fe-4S center of TMADH to the oxidized and semiquinone forms of
R237A ETF are +0.16 and +0.15
eV, respectively. Therefore, the equilibrium position for electron
transfer is shifted toward oxidized ETF and reduced TMADH. Using (i)
the robust engineering principles of Dutton and colleagues (27) for the
design of electron transfer proteins and (ii) the known electron
tunnelling distance of ~12 Å from the 4Fe-4S center to ETF (28), we
were able to make these driving forces compatible with intrinsic
endergonic tunnelling rates in the region of
102-104 s
1. Using
these arguments, it is perhaps surprising that the rate of reduction of
R237A ETF is severely compromised. However, as noted in our studies
of electron transfer from TMADH to ETF (28), the structural
rearrangement of ETF to form geometries compatible with electron
transfer in an "induced fit" process with TMADH can become
rate-limiting (especially with mutant forms of TMADH) for electron
transfer. The structural changes on complex formation can be followed
conveniently by difference spectroscopy studies of the wild-type
complex (18). For the wild-type complex, these spectral changes occur
rapidly, i.e. within the mixing time (<10 s). Similar
studies with native TMADH and the
R237A ETF also reveal that
spectral changes accompany complex formation (Fig. 6). However, the time course for
development of these spectral perturbations is protracted (~5 h),
indicating that assembly of the productive electron transfer complex is
severely compromised as a result of mutating
Arg-237. These
observations thus illustrate that the rate of rearrangement of ETF to
form the electron transfer complex, and not the intrinsic rate
(ket) of endergonic electron tunnelling, is
limiting for electron transfer from TMADH to
R237A ETF.
View larger version (25K):
[in a new window]
Fig. 5.
Reduction of wild-type and
R237A ETF by catalytic amounts of TMADH.
Reactions were performed under anaerobic conditions at 20 °C in 50 mM potassium phosphate buffer, pH 7.0. ETF concentration,
26 µM; trimethylamine concentration, 10 mM;
TMADH concentration, 0.1 and 1 µM for the wild-type and
R237A ETF, respectively. Main panel (
R237A ETF),
complete reduction was achieved only after 24 h of incubation.
Inset (wild-type (wt) ETF), complete reduction
was achieved in less than 5 min.
View larger version (27K):
[in a new window]
Fig. 6.
Spectral changes associated with the binding
of the TMADH: R237A ETF complex.
Main panel, difference absorption spectra (after
mixing-before mixing) of oxidized TMADH:
R237A ETF. Inset,
absorption change versus time after mixing of TMADH with
R237A ETF. Fitting of time-dependent absorption change
to a double exponential process produces observed rates for complex
assembly of 1.6 × 10
3
s
1 (fast phase, 10% total amplitude) and
8.5 × 10
5 s
1
(90% total amplitude change).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
520 mV
compared with
124 mV for FMN in aqueous solution (29). Detailed
potentiometric, mutagenesis, and structural studies have highlighted
the role of key hydrogen bonding interactions with the flavin N(5) (30,
31) and N(3) (32), the role of aromatic residues close to the flavin
isoalloxazine ring (33, 34), and conformational dynamics (35,
36) in modulating the potential of the one-electron couple in flavodoxins.
21 mV (11). The small difference in reduction
potentials between the human and Paracoccus proteins reflects the close similarity of the flavin surroundings apparent in
the crystallographic structures of these proteins (10, 11). Indeed,
based on detailed sequence alignments of ETFs from different species,
the flavin environment in ETFs is remarkably conserved across species
(11). On the basis of the conservation of
Arg-237 in all ETFs and
the wide variation in measured potentials, Frerman and co-workers (37)
have suggested that this residue is more likely to be essential to the
basic chemistry of ETFs, rather than a modulator of potentials. Our
mutagenesis work with M. methylotrophus ETF suggests that
this may not be the case, because
Arg-237 clearly plays a major role
in modulating the potential of the E'1 couple. However, the counterpart residue in human ETF (
Arg-249) is not sufficient to elevate the potential of the E'1
couple to the extent found in M. methylotrophus ETF. In
searching for other determinants of redox potential, notable
differences are apparent in the flavin environment of M. methylotrophus ETF and human ETF (Fig. 1). The human residues
Asn-259,
Thr-266,
Tyr-16, and
Pro-40 are exchanged for
lysine, serine, leucine, and glutamate, respectively, in M. methylotrophus ETF. Our data reveal that
R237A can more than account for the ~130-mV difference in the midpoint potential of E'1 couples of human and M. methylotrophus ETFs. However, human ETF also contains an arginine
at this position (as does P. denitrificans ETF). The
possibility arises, therefore, that exceptional stabilization of the
semiquinone in M. methylotrophus ETF may also require the flavin contact residues
Lys-247,
Ser-254,
Leu-13, and
Glu-37 in addition to
Arg-237.
Arg-249, the counterpart of
Arg-237 in M. methylotrophus ETF, has been exchanged for Lys. This exchange
leads to an ~60-mV destabilization of the flavin semiquinone (Table
I). This destabilization of the E'1 couple
probably reflects a shortening of the side chain and thus the movement
of the positive charge away from the dimethylbenzene subnucleus of the
FAD, potentially to form a salt bridge with
Asp-253. The same
mutation destabilizes the E'2 couple by 80 mV,
which contrasts with the >220-mV stabilization of the
E'2 couple in M. methylotrophus ETF
following introduction of the
R237A mutation (Table I). Mutation of
the other flavin contact residues,
Y16A and
D253A, in human ETF
produced only modest perturbations in the potentials of the
E'1 and E'2 couples (37). Our modeling suggests that the kinetic block of M. methylotrophus ETF arises (at least in part) from an interdomain
salt bridge between
Arg-237 and
Glu-37 (which cannot form in
human and P. denitrificans ETF, where the corresponding
residue is a Pro). This salt bridge might be broken upon reduction from
FAD semiquinone to FAD dihydroquinone and thus also provides a possible
explanation as to why reduction to the FAD dihydroquinone is relatively
facile in
R237A, unlike in the wild type. The ~200-mV
destabilization of the FAD semiquinone in
R237A could also be
explained in terms of this interdomain salt bridge; in
R237A the
(stabilizing) positive charge is removed from the si face of
the flavin, and a (destabilizing;
Glu-37) negative charge is
effectively introduced.
Arg-237 clearly plays a key role in
stabilizing the FAD semiquinone in M. methylotrophus ETF.
However, because an equivalent residue is present in other ETFs, other
factors must give rise to the exceptional stabilization of the FAD
semiquinone in M. methylotrophus ETF. The modeling suggests
a possible explanation for this; the positive charge on
Lys-247
(which is Asn in human and P. denitrificans ETF) lies <6 Å away from the si face of the pyrimidine subnucleus of FAD and could therefore stabilize the negative charge that builds up in the
pyrimidine subnucleus on reduction of the flavin to the anionic
semiquinone. The suggested role of
Lys-247 in the exceptional
stabilization of the FAD semiquinone will need to be explored in future work.
Arg-237 and
Glu-37, a
key restraint on domain II motion may be removed. This should enable
R237A ETF to more readily explore these alternative conformations,
thus perturbing the distribution between the different ETF conformers.
The
R237A ETF is clearly less kinetically restricted on being
reduced to the dihydroquinone level, supporting this hypothesis.
Arg-237 and possible interactions between
the 4'-hydroxyl and flavin N1 atom in providing a kinetic block on full
reduction to the dihydroquinone will need to be explored in future
work. However, it is clear from the analyses presented in this paper
that mutation of
Arg-237 to Ala goes a long way in relieving the
kinetic limitation on full reduction of M. methylotrophus
ETF.
Arg-237 in M. Methylotrophus ETF results in an ~200-mV destabilization of the
E'1 couple of the protein-bound FAD, which is an
unprecedented large perturbation in the midpoint reduction potential of
a flavin redox center facilitated by a single point mutation. Exchange
of this arginine residue partially removes a kinetic block and causes a
large elevation (>+280 mV) of the E'2 couple,
resulting in our ability to reduce the
R237A ETF completely
to the dihydroquinone (in the presence of mediators) with relatively
little formation of the anionic semiquinone that becomes fully
populated in wild-type ETF titrations. Our work underlines the unusual
properties of ETF and the role of a single residue near the flavin in
controlling the dynamic and redox properties of the molecule.
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FOOTNOTES |
---|
* This work was funded by grants from the Biotechnology and Biological Sciences Research Council (to M. J. S. and N. S. S.) and the Lister Institute of Preventive Medicine (to N. S. S.) and is also a contribution from the Edinburgh Protein Interaction Center funded by the Wellcome Trust.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.
¶ Present address: Dept. of Biochemistry, University of Leicester, University Rd., Leicester LE1 7RH, United Kingdom.
A Royal Society University Research Fellow.
§§ A Lister Institute Research Professor. To whom correspondence should be addressed. Tel.: 44 116 223 1337; Fax: 44 116 252 3369; E-mail: nss4@le.ac.uk.
Published, JBC Papers in Press, April 2, 2001, DOI 10.1074/jbc.M010853200
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ABBREVIATIONS |
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The abbreviations used are: ETF, electron-transferring flavoprotein; TMADH, trimethylamine dehydrogenase.
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REFERENCES |
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1. | Thorpe, C. (1991) in Chemistry and Biochemistry of Flavoenzymes (Muller, F., ed), Vol. II , pp. 471-486, CRC Press, Inc., Boca Raton, FL |
2. | Weidenhaupt, M., Rossi, P., Beck, C., Fischer, H. M., and Hennecke, H. (1996) Arch. Microbiol. 165, 169-178[CrossRef][Medline] [Order article via Infotrieve] |
3. | Husain, M., and Steenkamp, D. J. (1985) J. Bacteriol. 163, 709-715[Medline] [Order article via Infotrieve] |
4. | Steenkamp, D. J., and Gallup, M. (1978) J. Biol. Chem. 253, 4086-4089[Abstract] |
5. | Eichler, K., Buchet, A., Bourgis, F., Kleber, H. P., and Mandrand-Berthelot, M. A. (1995) J. Basic Microbiol. 35, 217-227[Medline] [Order article via Infotrieve] |
6. | Tsai, M. H., and Saier, M. H., Jr. (1995) Res. Microbiol. 146, 397-404[CrossRef][Medline] [Order article via Infotrieve] |
7. | Earl, C. D., Ronson, C. W., and Ausubel, F. M. (1987) J. Bacteriol. 169, 1127-1136[Medline] [Order article via Infotrieve] |
8. |
Whitfield, C. D.,
and Mayhew, S. G.
(1974)
J. Biol. Chem.
249,
2801-2810 |
9. | Sato, K., Nishina, Y., and Shiga, K. (1993) J. Biochem. (Tokyo) 114, 215-222[Abstract] |
10. |
Roberts, D. L.,
Frerman, F. E.,
and Kim, J.-J. P.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14355-14360 |
11. | Roberts, D. L., Salazar, D., Fulmer, J. P., Frerman, F. E., and Kim, J. J. (1999) Biochemistry 38, 1977-1989[CrossRef][Medline] [Order article via Infotrieve] |
12. | Duplessis, E. R., Rohlfs, R. J., Hille, R., and Thorpe, C. (1994) Biochem. Mol. Biol. Int. 32, 195-199[Medline] [Order article via Infotrieve] |
13. | Gorelick, R. J., Mizzer, J. P., and Thorpe, C. (1982) Biochemistry 21, 6936-6942[Medline] [Order article via Infotrieve] |
14. | Husain, M., and Steenkamp, D. J. (1983) Biochem. J. 209, 541-545[Medline] [Order article via Infotrieve] |
15. | Watmough, N. J., Kiss, J., and Frerman, F. E. (1992) Eur. J. Biochem. 205, 1089-1097[Abstract] |
16. | Davidson, V. L., Husain, M., and Neher, J. W. (1986) J. Bacteriol. 166, 812-817[Medline] [Order article via Infotrieve] |
17. | Byron, C. M., Stankovich, M. T., Husain, M., and Davidson, V. L. (1989) Biochemistry 28, 8582-8587[Medline] [Order article via Infotrieve] |
18. |
Jang, M.-H.,
Scrutton, N. S.,
and Hille, R.
(1999)
J. Biol. Chem.
275,
12546-12552 |
19. | Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231-236 |
20. |
Jones, M.,
Basran, J.,
Sutcliffe, M. J.,
Gunter Grossmann, J.,
and Scrutton, N. S.
(2000)
J. Biol. Chem.
275,
21349-21354 |
21. | Wilson, E. K., Huang, L., Sutcliffe, M. J., Mathews, F. S., Hille, R., and Scrutton, N. S. (1997) Biochemistry 36, 41-48[CrossRef][Medline] [Order article via Infotrieve] |
22. | Barber, M. J., Pollock, V., and Spence, J. T. (1988) Biochem. J. 256, 657-659[Medline] [Order article via Infotrieve] |
23. | White, S. A., Mathews, F. S., Rohlfs, R. J., and Hille, R. (1994) J. Mol. Biol. 240, 265-266[CrossRef][Medline] [Order article via Infotrieve] |
24. | Dutton, P. (1978) Methods Enzymol. 54, 422-435 |
25. | Daff, S. N., Chapman, S. K., Turner, K. L., Holt, R. A., Govindaraj, S., Poulos, T. L., and Munro, A. W. (1997) Biochemistry 36, 13816-13823[CrossRef][Medline] [Order article via Infotrieve] |
26. | Muller, F., Mayhew, S. G., and Massey, V. (1973) Biochemistry 12, 4654-4662[Medline] [Order article via Infotrieve] |
27. | Page, C. C., Moser, C. C., Chen, X. X., and Dutton, P. L. (1999) Nature 402, 47-52[CrossRef][Medline] [Order article via Infotrieve] |
28. | Basran, J., Chohan, K. K., Sutcliffe, M. J., and Scrutton, N. S. (2000) Biochemistry 39, 9188-9200[CrossRef][Medline] [Order article via Infotrieve] |
29. | Mayhew, S. G., and Tollin, G. (1992) in Chemistry and Biochemistry of Flavoenzymes (Muller, F., ed) , pp. 389-426, CRC Press, Inc., Boca Raton, FL |
30. | Chang, F. C., and Swenson, R. P. (1999) Biochemistry 38, 7168-7176[CrossRef][Medline] [Order article via Infotrieve] |
31. | O'Farrell, P. A., Walsh, M. A., McCarthy, A. A., Higgins, T. M., Voordouw, G., and Mayhew, S. G. (1998) Biochemistry 37, 8405-8416[CrossRef][Medline] [Order article via Infotrieve] |
32. | Bradley, L. H., and Swenson, R. P. (1999) Biochemistry 38, 12377-12386[CrossRef][Medline] [Order article via Infotrieve] |
33. | Lostao, A., Gomez-Moreno, C., Mayhew, S. G., and Sancho, J. (1997) Biochemistry 36, 14334-14344[CrossRef][Medline] [Order article via Infotrieve] |
34. | Swenson, R. P., and Krey, G. D. (1994) Biochemistry 33, 8505-8514[Medline] [Order article via Infotrieve] |
35. | Kasim, M., and Swenson, R. P. (2000) Biochemistry 39, 15322-15332[CrossRef][Medline] [Order article via Infotrieve] |
36. | Ludwig, M. L., Pattridge, K. A., Metzger, A. L., Dixon, M. M., Eren, M., Feng, Y., and Swenson, R. P. (1997) Biochemistry 36, 1259-1280[CrossRef][Medline] [Order article via Infotrieve] |
37. | Dwyer, T., Zhang, L., Muller, M., Marrugo, F., and Frerman, F. (1999) Biochim. Biophys. Acta 1433, 139-152[Medline] [Order article via Infotrieve] |
38. | Dwyer, T. M., Mortl, S., Kemter, K., Bacher, A., Fauq, A., and Frerman, F. E. (1999) Biochemistry 38, 9735-9745[CrossRef][Medline] [Order article via Infotrieve] |
39. | Massey, V. (1991) in Flavins and Flavoproteins (Curti, B. , Ronchi, S. , and Zanetti, G., eds) , pp. 59-66, Walter de Gruyter & Co., Berlin |
40. | Husain, M., Stankovich, M. T., and Fox, B. G. (1984) Biochem. J. 219, 1043-1047[Medline] [Order article via Infotrieve] |