(Received for publication, May 9, 1995; and in revised form, August 2, 1995)
From the
The kinetics of electron transfer between trimethylamine
dehydrogenase (TMADH) and its physiological acceptor, electron
transferring flavoprotein (ETF), has been studied by static and
stopped-flow absorbance measurements. The results demonstrate that
reducing equivalents are transferred from TMADH to ETF solely through
the 4Fe/4S center of the former. The intrinsic limiting rate constant (k) and dissociation constant (K
) for electron transfer from the
reduced 4Fe/4S center of TMADH to ETF are about 172 s
and 10 µM, respectively. The reoxidation of fully
reduced TMADH with an excess of ETF is markedly biphasic, indicating
that partial oxidation of the iron-sulfur center in 1-electron reduced
enzyme significantly reduces the rate of electron transfer out of the
enzyme in these forms. The interaction of the two unpaired electron
spins of flavin semiquinone and reduced 4Fe/4S center in 2-electron
reduced TMADH, on the other hand, does not significantly slow down the
electron transfer from the 4Fe/4S center to ETF. From a comparison of
the limiting rate constants for the oxidative and reductive
half-reactions, we conclude that electron transfer from TMADH to ETF is
not rate-limiting during steady-state turnover. The overall kinetics of
the oxidative half-reaction are not significantly affected by high salt
concentrations, indicating that electrostatic forces are not involved
in the formation and decay of reduced TMADH-oxidized ETF complex.
Trimethylamine dehydrogenase (TMADH; ()EC 1.5.99.7)
isolated from the methylotrophic bacterium W
A
is a homodimer of molecular weight of 166,000, with each subunit
containing a covalently bound 6-cysteinyl FMN coenzyme and a 4Fe/4S
(ferredoxin-type) iron-sulfur
center(1, 2, 3, 4) . TMADH also
possesses 1 equivalent of tightly bound ADP/monomer, although the
function of this cofactor remains unknown(5) . The enzyme
catalyzes the oxidative demethylation of trimethylamine to
dimethylamine and formaldehyde, passing the pair of reducing
equivalents thus obtained individually to its physiological oxidant, an
electron-transferring flavoprotein (ETF), which becomes reduced to the
level of the (anionic)
semiquinone(4, 6, 7, 8) . The ETF
from W
A
has been shown to be an
dimer with molecular weight of 77,000 and contains 1 mol of FAD and
AMP/mol of protein; the role of AMP is unclear.
Complete reduction
of TMADH requires 3 electrons/subunit, 2 for full reduction of the FMN,
and a 3rd for reduction of the iron-sulfur center. When TMADH is
reduced to the level of 2 electrons/subunit, there are two possible
distributions of reducing equivalents: 1) fully reduced FMN with
oxidized iron-sulfur and 2) flavin semiquinone with reduced iron-sulfur
center. At pH 7.0, the former distribution is favored by a factor of
approximately 2:1. Furthermore, although some TMADH possesses flavin semiquinone and reduced iron-sulfur center at pH
7.0, the magnetic moments of the unpaired spins do not interact as is
the case when TMADH is reduced by excess substrate, or by sodium
dithionite at high pH or in the presence of the inhibitor
tetramethylammonium
chloride(6, 9, 10, 11) . This
spin-interacting form exhibits a unique EPR signal that includes
half-field features and is not simply the sum of the signals for flavin
semiquinone and reduced iron-sulfur center; we designate this
spin-interacting form as TMADH
*.
Previous
freeze-quench studies have demonstrated that when TMADH*
is mixed with ETF
, the EPR signal arising from the
spin-interacting state is lost within a few milliseconds with no
concomitant appearance of that for the reduced iron-sulfur center, as
would be expected if electrons are transferred from the
flavosemiquinone of TMADH
* to ETF
to give
enzyme possessing oxidized flavin and reduced 4Fe/4S center (11) . On the basis of these results, it has been proposed that
electrons are transferred from the iron-sulfur center of TMADH to
ETF
(11) , but direct evidence has been lacking.
The present work provides direct evidence that electron transfer to ETF
takes place exclusively via the iron-sulfur center of TMADH and
determines the intrinsic rate constant for electron transfer and
dissociation constant using stopped-flow rapid mixing technique. The
results are incorporated into a comprehensive kinetic mechanism for the
reaction of TMADH.
Figure 1:
Panel A, TMADH
optical spectra. The spectra shown are for oxidized enzyme (solidline), 2-electron reduced enzyme (dashedline), and fully reduced enzyme (dottedline) at pH 7.0. Enzyme was reduced with titanium citrate
to a level of 2 or 3 electrons/subunit. Panel B, ETF optical
spectra. The spectra shown are for oxidized ETF (solidline) and dithionite reduced ETF (dottedline) in 50 mM potassium phosphate, pH 7.0. Panel C, optical spectra observed before and after mixing of
fully reduced TMADH with oxidized ETF. Dithionite fully reduced TMADH
(8.2 µM) was mixed anaerobically with 50 µM of oxidized ETF in 50 mM phosphate buffer, pH 7.0, in a
split cell (see ``Materials and Methods''). The solidline is the spectrum recorded before mixing and the
dotted line is the spectrum taken after mixing. Panel D,
optical spectra observed before and after mixing of 2-electron reduced
TMADH with oxidized ETF. TMADH (14 µM) reduced with
titanium citrate to the level of 2 eq/subunit was mixed anaerobically
with 42 µM oxidized ETF in 50 mM phosphate
buffer, pH 7.0, in a split cell. The solidline is
the spectrum recorded before mixing, and the dottedline is the spectrum taken after mixing. Panel E, theoretical
and experimental difference spectra for the reaction of TMADH with ETF
. The theoretical difference
spectrum (dottedline) was obtained by adding the
difference spectrum for reoxidation of 4.1 µM
TMADH
(panelA) to the difference
spectrum for reduction of 12.3 µM ETF
(panelB). The experimental difference spectrum (solid line) was obtained by subtracting the solidline in panelC from the dottedline in panelC.
Comparable results to those described above are obtained when only
partially reduced enzyme was used. When 1.0 ml of 14 µM TMADH in the non-spin-interacting state (generated
by titration with Ti
citrate to the level of 2
reducing equivalents/subunit at pH 7.0) is mixed with 1.0 ml of 42
µM ETF
, the spectral change shown in Fig. 1D is observed and found to be quantitatively
consistent with the reduction of ETF
and the reoxidation
of TMADH
in the ratio of 2:1. When TMADH
in
the spin-interacting state (generated by reduction with 1 equivalent of
trimethylamine in the presence of 3 mM tetramethyl ammonium
chloride) is mixed anaerobically with ETF
at pH 7.0 in a
split cell, the spectral change is again consistent with the reduction
of ETF
and the reoxidation of the spin interacting state
of TMADH
in the ratio 2:1. These results demonstrate that
ETF is able to fully reoxidize TMADH.
Figure 2:
Panel A,
phenylhydrazine-inactivated TMADH optical spectra. The spectra shown
are for oxidized enzyme (solidline) and dithionite
reduced enzyme (dottedline) in 50 mM phosphate buffer, pH 7.0. Panel B, optical spectra
observed before and after mixing of reduced phenylhydrazine-inactivated
TMADH with oxidized ETF. 1 ml of 12 µM phenylhydrazine-inactivated TMADH in 50 mM phosphate
buffer, pH 7.0, was reduced with dithionite and then placed in one side
of an anaerobic split cell. 1 ml of 34 µM anaerobic
oxidized ETF in 50 mM phosphate buffer, pH 7.0, was placed in
the other side of the split cell. The solidline is
the spectrum recorded before mixing of phenylhydrazine-inactivated
TMADH with oxidized ETF, and the dottedline is the
spectrum recorded after mixing. Panel C, theoretical and
experimental difference spectra for the reaction of reduced
phenylhydrazine-inactivated TMADH with ETF. The
theoretical difference spectrum (dottedline) was
obtained by adding the difference spectrum for reoxidation of 6
µM reduced phenylhydrazine-inactivated TMADH (panelA) to the difference spectrum for reduction of 6
µM ETF
. The experimental difference spectrum (solidline) was obtained by subtracting the solidline in panelB from the dottedline in panelB.
Figure 3:
Deconvolution of the difference spectrum
for oxidized and fully reduced TMADH. The difference spectrum for
oxidized and reduced 4Fe/4S center (solidline) is
obtained by subtracting the spectrum for reduced
phenylhydrazine-inactivated TMADH from that for oxidized inactivated
TMADH. The difference spectrum for oxidized and reduced TMADH (dashedline) is obtained by subtracting the
spectrum for fully reduced TMADH from that for oxidized TMADH. The
difference spectrum for the enzyme FMN and FMNH (dottedline) is obtained by subtracting the difference spectrum
for 4Fe/4S center from that for TMADH.
Treatment of TMADH with 3 mM ferricenium hexafluorophosphate at pH 10 for 4 h at room
temperature has been found empirically to give an iron-sulfur center
that is EPR-active. ()The EPR spectrum of the enzyme thus
generated is found to superficially resemble that given by the oxidized
form of various high potential iron proteins(20) . The
integrated spin intensity of this signal indicates that the iron-sulfur
center is quantitatively converted to this paramagnetic state. Since
there was no loss of iron associated with generation of this EPR-active
species (as one would expect if the signal arose from formation of a
3Fe/4S center), we tentatively conclude that the procedure results in
the 1-electron oxidation of the iron-sulfur center to a level
corresponding to that for oxidized high potential iron proteins.
Regardless of the nature of this oxidation product, the significant
aspect with regard to the present work is that the procedure renders
the iron-sulfur center of TMADH redox-inert in that treatment with
trimethylamine for 30 min does not reduce the intensity of the new EPR
signal. (
)It is found, however, that trimethylamine is still
able to react with and reduce the enzyme FMN, but the reduced enzyme
thus generated was not able to reduce ETF
, even after
prolonged incubation. These results demonstrate the flavin center of
ferricenium-treated TMADH remains catalytically competent, but that
rendering the iron-sulfur center redox-inert prevents reoxidation of
the reduced flavin by ETF.
Figure 4:
Time courses observed for the reactions of
reduced phenylhydrazine-inactivated TMADH and 3-electron reduced TMADH
with oxidized ETF. Absorbance changes observed at 370 nm (closedtriangles) and 440 nm (closedcircles)
after mixing in a stopped-flow apparatus are plotted versus time. The reaction conditions are 50 mM potassium
phosphate, pH 7.0, 25 °C. The symbols represent the data points,
and the solidlines represent fits of the data to
exponentials of the form A(t) =
A
exp(-k
t)
where
A and k
represent the
absorbance change and observed rate constant exhibited by the nth kinetic phase, respectively. PanelA,
reaction of dithionite reduced phenylhydrazine-inactivated TMADH with
ETF
. The concentrations after mixing are:
[inactivated TMADH] = 3 µM,
[ETF] = 58 µM. Data are fitted to the sum
of two exponentials: k
= 141
s
and k
= 3
s
. The observed rate constants for each kinetic
phase are independent of observation wavelength. PanelB, reaction of TMADH
with ETF
.
After mixing, [TMADH] = 1 µM,
[ETF] = 45 µM. Only every 10th point is
shown to allow visualization of the fitted curves. Data at 370 nm are
fitted to the sum of two exponentials: k
= 138 s
and k
= 12 s
. Data at 440 nm are fitted to a
single exponential: k = 12
s
.
Figure 5:
Double-reciprocal plot of the observed
rate constants versus ETF concentration. All reactions were measured in
a stopped-flow apparatus at 370 nm and 25 °C. The buffer was 50
mM potassium phosphate, pH 7.0. The opencircles represent the data for the fast phase of the reaction of
3-electron reduced TMADH with ETF. The enzyme concentration after
mixing was 1.0 µM. The fit of the data gives k = 172 s
and K
= 10 µM. The opensquares represent the data for the reaction of
phenylhydrazine-inactivated TMADH with ETF. The concentration of
inactivated TMADH after mixing was 3.0 µM. The fit of the
data gives k
= 173 s
and K
= 16 µM.
The filledcircles represent the data for the fast
phase of TMADH
in the spin-interacting state with ETF.
The enzyme concentration after mixing was 2.3 µM. The fit
of the data gives k
= 149 s
and K
= 39 µM.
The filledsquares represent the data for the fast
phase of the reaction of TMADH
in non-spin-interacting
state with ETF. The concentration of TMADH after mixing was 2.1
µM. The fit of the data gives k
= 157 s
and K
= 24 µM.
Figure 6:
Static and kinetic difference spectra. PanelA, the solidline is the
static difference spectrum calculated from the spectrum taken after
mixing of TMADH (14 µM) with ETF
(42 µM) at pH 7.0 minus the spectrum recorded before
mixing (see Fig. 1D). The closed circles represent the kinetic data obtained by mixing TMADH
(14 µM) with ETF
(37 µM)
in 50 mM phosphate buffer, pH 7.0, at 25 °C in a
stopped-flow apparatus. PanelB, the solidline is the static difference spectrum calculated from
the spectrum taken after mixing of dithionite-reduced
phenylhydrazine-inactivated TMADH (12 µM) with ETF
(34 µM) in 50 mM phosphate buffer, pH 7.0,
minus the spectrum recorded before mixing (see Fig. 2B). The closedcircles represent the kinetic data obtained by mixing reduced
phenylhydrazine-inactivated TMADH (12 µM) with ETF
(36 µM) in 50 mM phosphate buffer, pH 7.0,
in a stopped-flow apparatus at 25 °C.
The temperature dependence of the rate of electron
transfer from the 4Fe/4S center of TMADH to ETF was studied by reacting
dithionite-reduced phenylhydrazine-inactivated TMADH with ETF at 5, 15, 25, and 35 °C. The observed rate constants were
temperature dependent and the Arrhenius plot is linear (not shown),
giving activation energy of 12.8 kcal/mol.
The reaction of fully
reduced native TMADH with ETF also exhibits two kinetic phases,
although in this case the extent of the spectral change associated with
the slow phase (approximately 67%) appears to be kinetically
significant. Fig. 4B shows kinetic transients obtained
on mixing TMADH with excess ETF
at pH 7.0 in
a stopped-flow spectrophotometer. The transient observed at 370 nm
consists of both phases, while that observed at 440 nm contains only
the slow phase. The observed rate constants for each kinetic phase at a
given set of reaction conditions are found to be independent of
observation wavelength over the range of 300-600 nm. At 370 nm,
the fast phase accounts for about one third of the total absorbance
change and the observed rate constant is dependent on the ETF
concentration; a double-reciprocal plot of k
for
the fast phase versus ETF concentration is linear (Fig. 5, opencircles) and the fit of the data
gives k
= 172 s
and K
= 10 µM, in good agreement
with the results using phenylhydrazine-inactivated enzyme. The observed
rate constant for the slow phase is also ETF concentration dependent
and the double-reciprocal plot of k
versus ETF concentration is linear (not shown), giving k
= 16 s
and K
= 9.9 µM. The overall kinetics are consistent
with the rapid removal of the first reducing equivalents from fully
reduced TMADH, followed by the much slower removal of the second and
third equivalents from the 2-electron reduced enzyme thus generated
(owing to an unfavorable distribution of the reducing equivalents in
TMADH
and TMADH
, see
``Discussion'').
It is of interest to determine whether
the rate constant for the reaction of reduced iron-sulfur center in
TMADH with ETF
is dependent on whether the
iron-sulfur center existed in a strong magnetic interaction with the
flavin site. As in the case of fully reduced enzyme, the reaction of
TMADH
in the non-spin-interacting state with ETF
is biphasic (data not shown). The fast phase accounts for about
30% of the total absorbance change, and the observed rate constant is
dependent on the ETF concentration; a double-reciprocal plot of k
for the fast phase versus ETF
concentration is linear (Fig. 5, filledsquares), and the fit of the data gives k
= 157 s
and K
= 24 µM. The observed rate
constant for the slow phase is approximately 10 s
and is independent of [ETF] under pseudo first-order
conditions. In this experiment, approximately 40% of the TMADH
exists initially with an electron distribution possessing flavin
semiquinone and reduced iron-sulfur center, but in which the two
unpaired spins are not interacting to any detectable
extent(22) . When the experiment is repeated using
TMADH
in the spin-interacting state (generated by
reduction of the enzyme with 1 equivalent of trimethylamine in the
presence of 3 mM tetramethyl ammonium chloride), the reaction
again exhibits two kinetic phases (data not shown). The fast phase
accounts for about 70% of the total absorbance change and the observed
rate constant is ETF concentration dependent, with k
and K
of 149 s
and 39
µM, respectively (Fig. 5, filledcircles). The rate constant for the slow phase, which
accounts for approximately 30% of the total absorbance change, is about
4 s
and independent of ETF concentration.
The present results indicate that when TMADH is treated with phenylhydrazine, rendering the FMN redox-inert(13) , the iron-sulfur center can be reduced by dithionite and reoxidized by ETF. Similarly, when TMADH is treated with ferricenium hexafluorophosphate at high pH, oxidizing the iron-sulfur center to a paramagnetic but redox-inert state, the FMN can be reduced by trimethylamine but cannot be reoxidized by ETF. These results strongly suggest that reducing equivalents introduced into TMADH at the flavin site in the course of turnover are transferred to ETF exclusively via the iron-sulfur center. This is consistent with the interpretation of previous kinetic results, which have also implicated the iron-sulfur center as the site of the oxidative half-reaction of TMADH(11) .
The reaction of ETF
with reduced, phenylhydrazine-inactivated enzyme gives the intrinsic
kinetic parameters for the reaction of ETF with the fully reduced
iron-sulfur center without the complication of subsequent electron
transfer from the flavin and further reduction of ETF. The limiting
rate constant of 173 s for electron transfer from
the reduced iron-sulfur center to ETF is much faster than the
rate-limiting step in the reductive half-reaction (product
dissociation, with a rate constant of 3.5 s
; (9) ), so electron transfer from the iron-sulfur center of
TMADH to ETF is not rate-limiting during steady-state turnover. The
fact that both k
and K
(16
µM) are insensitive to high salt concentration indicates
that electrostatic forces are not involved in the formation and decay
of E
ETF
complex. The good
agreement between both k
and K
for the reoxidation of reduced, phenylhydrazine-inactivated TMADH
with ETF
and the fast phase of the reoxidation of fully
reduced enzyme support the conclusion that the former reaction
accurately represents the intrinsic reaction of enzyme possessing fully
reduced iron-sulfur center with ETF, and that reaction of the enzyme
flavin with phenylhydrazine does not significantly perturb the
iron-sulfur center.
The fast phase of the reaction of TMADH with ETF accounts for one third of the total absorbance change,
and k
is the same as that for the reaction of
phenylhydrazine-inactivated TMADH with ETF, indicating the fast phase
represents the removal of the first reducing equivalent from the
reduced 4Fe/4S center to give TMADH
. Because the slow
phase accounts for two thirds of the total absorbance change, it most
likely represents removal of the second and third equivalents from the
FMNH
of TMADH
steps which are not
kinetically resolved. Consistent with this interpretation is the good
agreement between the rate constants for the slow phases of the
reactions of ETF with TMADH
and TMADH
(16
s
and 10 s
, respectively). The
small rate constants for both slow phases are owing to the unfavorable
distribution of the reducing equivalents in TMADH
and
TMADH
. The distribution of the reducing equivalents
favors the enzyme form with FMNH
and oxidized 4Fe/4S in
TMADH
and with FMNH
and oxidized 4Fe/4S in
TMADH
. The reoxidation of fully reduced TMADH by ETF can
thus be summarized as shown in Fig. S1.
Figure S1: Scheme 1.
The fast phases of
the reactions of ETF with TMADH in non-spin-interacting
state and in spin-interacting state account for 30% and 70% of the
total absorbance change, respectively, presumably because only 40% of
the 4Fe/4S center in TMADH
in non-spin-interacting state
and all the 4Fe/4S center in TMADH
in spin-interacting
state are reduced. k
for the fast phases of the
two reactions are within experimental error (15-20%, (23) ) of the value for the reaction of
phenylhydrazine-inactivated TMADH with ETF. Thus, both fast phases of
the reaction of ETF with TMADH
in non-spin-interacting
state and in spin-interacting state represent the electron transfer
from the reduced 4Fe/4S center in TMADH
to ETF and the
rate of the electron transfer is independent of the reduction state of
the FMN. k
(149 s
) for the
fast phase of the reaction of TMADH
in the
spin-interacting state with ETF is identical, within experimental
error, to that for the fast phase of the reaction of TMADH
in non-spin-interacting state with ETF (157
s
). This indicates that formation of the
spin-interacting state does not significantly slow down the electron
transfer from the 4Fe/4S center to ETF and that binding of
tetramethylammonium chloride has little effect on the rate of electron
transfer from the iron-sulfur center of TMADH to ETF. As in the absence
of tetramethylammonium chloride, the slow phase represents the
oxidation of TMADH
, in which the sole reducing equivalent
resides primarily on the FMN center. The somewhat slower rate constant
for the slow phase of the reaction in the presence of
tetramethylammonium chloride (4 s
versus 10
s
in its absence) is consistent with the observation
that binding of tetramethylammonium chloride raises the FMN/FMNH
half-potential(19) , thereby further shifting the
oxidation-reduction equilibrium within TMADH
even further
toward flavin reduction and iron-sulfur oxidation and slowing the rate
of reaction with ETF.
Our kinetic results concerning the reaction of
the various reduced forms of TMADH with ETF can be incorporated into a
comprehensive kinetic mechanism for the turnover of TMADH with
trimethylamine and ETF. Because of the unusual situation where reducing
equivalents are introduced into the enzyme in pairs (at the FMN) and
removed one at a time (at the iron-sulfur center), coupled with the
ability of the enzyme to take up a total of 3 equivalents, the general
mechanism shown in Fig. S2must be considered. Two alternate
paths exist in which the enzyme alternates between: 1) oxidized and
2-electron reduced forms (the ``0/2 Cycle'' shown on the left
of Fig. S2, prevailing at low concentrations of trimethylamine)
and 2) between 1- and 3-electron reduced forms (the ``1/3
Cycle'' shown on the right, prevailing at high concentrations of
trimethylamine). The cycle in which the enzyme operates depends on the
fate of TMADH formed after reaction of oxidized enzyme
with 1 equivalent of trimethylamine. This species can react either with
ETF to give oxidized enzyme, or with substrate to give the
spin-interacting state, which subsequently leads to formation of fully
reduced enzyme. Ultimately which branch is favored under a given set of
experimental conditions depends on the relative concentrations of ETF
and TMA and their relative rates of reaction with enzyme.
Figure S2: Scheme 2.
The
reductive half-reaction of TMADH has been studied by using both
substrate trimethylamine and nonphysiological substrate,
diethylmethylamine(9, 10, 11, 23, 24) .
Initial reduction of the enzyme by trimethylamine occurs at the flavin
site and is very rapid (t 2 ms, [TMA] =
500 µM). Following this initial rapid reduction, two
slower kinetic phases (t are approximately 80 and 200 ms,
respectively) are observed. The rate constant for the slowest phase is
approximately equal to k
(9, 10, 11, 24) . By using the
non-physiological substrate, diethylmethylamine, it has been
demonstrated that product release and the binding of the second
substrate molecule to the 2-electron reduced enzyme is the
rate-limiting step(23) . Intramolecular electron transfer
within TMADH
has been studied using a pH jump technique
and intramolecular equilibration of reducing equivalents is fast (k
200 s
)(25) .