(Received for publication, September 13, 1994; and in revised form, March 6, 1995)
From the
The pH dependence of static optical/EPR spectra of
trimethylamine dehydrogenase reduced to the level of two equivalents
(TMADH) has been examined and indicates the existence of
three different states for this iron-sulfur flavoprotein. At pH 6,
TMADH
exists principally in a form possessing flavin
mononucleotide hydroquinone, with its iron-sulfur center oxidized. At
pH 8, the enzyme principally contains flavin mononucleotide semiquinone
and reduced iron-sulfur, but despite the proximity of the two centers
to one another, their magnetic moments do not interact. At pH 10,
TMADH
exhibits the EPR spectrum that is diagnostic of a
previously characterized spin-interacting state in which the magnetic
moments of the flavin semiquinone and reduced iron-sulfur center are
strongly ferromagnetically coupled. The kinetics of the interconversion
of these three states have been investigated using a pH jump technique
in both H
O and D
O. The observed kinetics are
consistent with a reaction mechanism involving sequential
protonation/deprotonation and intramolecular electron transfer events.
All reactions studied show a normal solvent kinetic isotope effect.
Proton inventory analysis indicates that at least one proton is
involved in the reaction between pH 6 and 8, which principally controls
intramolecular electron transfer, whereas at least two protons are
involved between pH 8 and 10, which principally control formation of
the spin-interacting state. The results of these and previous studies
indicate that for TMADH
, between pH 10 and 6, at least
three protonation/deprotonation events are associated with
intramolecular electron transfer and formation of the spin-interacting
state, with estimated pK
values of 6.0,
8.0, and
9.5. These pK
values are
attributed to the flavin hydroquinone, flavin semiquinone, and an
undesignated basic group on the protein, respectively.
Trimethylamine dehydrogenase (TMADH; ()EC 1.5.99.1)
as isolated from bacterium W
A
is a 166-kDa
iron-sulfur flavoprotein. The enzyme consists of two identical,
catalytically independent subunits, (
)each containing a
covalently bound 6-cysteinyl FMN coenzyme and a 4Fe/4S (bacterial
ferredoxin-type) iron-sulfur
center(1, 2, 3, 4) . During
turnover, trimethylamine dehydrogenase is reduced by trimethylamine at
the FMN site (7, 8, 9) and is believed to be
oxidized at the iron-sulfur center, in vivo, by an
electron-transferring flavoprotein (5, 6) . (
)Intramolecular transfer of reducing equivalents from FMN
to the iron-sulfur center thus appears to be a necessary step in the
catalytic cycle. Full reduction of trimethylamine dehydrogenase
requires the uptake of three reducing equivalents, two for full
reduction of the FMN, and the third for reduction of the iron-sulfur
center. When reduced to the level of two equivalents/active site, an
equilibrium distribution of reducing equivalents between the two
centers exists, which is dictated by the relative reduction potentials
of the FMN and 4Fe/4S center. Two possible electron distributions
within the enzyme reduced by only two equivalents (TMADH
)
exist: 1) flavin hydroquinone and oxidized iron-sulfur center and 2)
flavin semiquinone and reduced iron-sulfur center. The latter
distribution has been seen with trimethylamine dehydrogenase reduced by
excess substrate, with enzyme reduced by sodium dithionite in the
presence of the inhibitor tetramethylammonium chloride, and with
dithionite-reduced enzyme at pH
10(6, 7, 8, 9, 10, 11) .
In each case, interaction of the magnetic moments of the unpaired
electrons of the flavin semiquinone and reduced iron-sulfur center
gives rise to a spin-interacting state, which is distinguished by a
complex EPR signal centered near g = 2 and an unusually
intense half-field (g
4) signal(12) . It has
been shown that this spin-interacting state forms in the absence of any
substantive protein conformational changes(13) .
Reductive optical/EPR titrations of trimethylamine dehydrogenase with sodium dithionite have shown that the equilibrium distribution of reducing equivalents between the FMN and 4Fe/4S center in partially reduced enzyme is dependent on pH(11) . Formation of fully reduced flavin with oxidized iron-sulfur is favored at low pH, whereas formation of flavin semiquinone and reduced iron-sulfur is preferred at high pH. The absorption spectrum of partially reduced enzyme exhibits changes that reflect this pH dependence, making it possible to follow the kinetics of intramolecular electron transfer with a stopped-flow rapid mixing apparatus by using a pH jump technique(11) . In these experiments, partially reduced enzyme is prepared in a weakly buffered (e.g. 10 mM) solution at a known initial pH and then rapidly mixed with a strongly buffered (e.g. 100 mM) solution at a different final pH. The initial equilibrium distribution of reducing equivalents within the enzyme (determined by the initial pH) is thus rapidly perturbed, permitting the kinetics of the approach of the system to the new equilibrium position (determined by the final pH) to be followed spectrophotometrically(14) .
Both electron transfer (from the flavin hydroquinone to the oxidized
iron-sulfur center) and formation of the spin-interacting state occur
in the course of the catalytic cycle of trimethylamine dehydrogenase.
The present studies have been undertaken to further examine the role of
protonation/deprotonation events in each of these two processes. We
find that the behavior of the enzyme is best described by a model in
which electron transfer and protonation/deprotonation events are
treated as independent equilibria. In addition, evidence is presented
that formation of the spin-interacting state of trimethylamine
dehydrogenase is governed by an ionization having a
pK of
9.5.
Earlier studies have shown that the bisulfite product of dithionite
oxidation reacts with oxidized trimethylamine dehydrogenase at pH
7 (11) . As an alternative to dithionite, low pH enzyme samples
used in optical/EPR measurements were reduced with titanium citrate
solutions (see below). This method of reduction is inappropriate for
the preparation of pH jump samples; however, since titanium citrate
requires strongly buffered (0.1-0.2 M) citrate solutions
to remain in solution. The bisulfite adduct problem was avoided in the
earlier pH jump study by first reducing the enzyme in 1 mM borate buffer, pH 10, and then bringing the solution to 10 mM phosphate at the desired initial pH by the addition of
concentrated phosphate buffer(11) . However, for the purposes
of this study, the complexity of this procedure would likely lead to
H
O contamination of D
O solutions and was not
used. Since formation of the bisulfite adduct renders the FMN
redox-inert, any enzyme molecules that have reacted with bisulfite
(estimated to be no greater than 10-20% in any case) are assumed
not to contribute to the absorbance change on the timescale of the
pH(D) jump experiment and merely add to the total background
absorbance. Any substantial breakdown of a sulfite adduct under the
present experimental conditions would be expected to give rise to
extremely slow spectral changes. The absence of such slow spectral
changes in the transients observed in the course of the work presented
here indicates that sulfite adduct formation presents a negligible
problem in the present studies.
Figure 1:
pH dependence of TMADH optical spectra. The spectra shown are for oxidized enzyme at pH
7.0 (upper solid line), enzyme reduced by dithionite to 2
equivalents/subunit at pH 10.0(- - -), pH 8.0(- - -
-), and by titanium citrate at pH 6.0 (
), and
enzyme fully reduced by dithionite at pH 7.0 (lower solid
line). The two-electron reduced sample at pH 10.0 was prepared by
titration with dithionite solution in 0.1 M borate buffer, pH
10.0, containing 0.5 µM benzyl viologen. The two-electron
reduced sample at pH 8.0 was prepared by titration with dithionite to
the level of 2 equivalents/subunit in 0.1 M sodium
pyrophosphate, pH 8.0, containing 0.5 µM benzyl viologen.
The two-electron reduced sample at pH 6.0 was prepared by titration
with titanium citrate solution in 0.1 M potassium phosphate
buffer, pH 6.0, containing 0.5 µM benzyl viologen.
Titanium citrate was used to prevent the formation of bisulfite-TMADH
complexes at low pH. The kinetically determined absorption spectrum of
the first intermediate formed in the course of the reaction of
trimethylamine dehydrogenase with diethylmethylamine, exhibiting the
spectrum of reduced flavin and oxidized iron-sulfur center, is shown (open circles) for
comparison(11) .
Figure 2:
EPR spectra of TMADH at pH
10.0 and 8.0. Enzyme in 0.1 M potassium borate, pH 10.0, or
0.1 M sodium pyrophosphate, pH 8.0, buffer containing 0.5
µM benzyl viologen was reduced with dithionite to 2
equivalents/subunit. EPR parameters were as follows: microwave
frequency, 9.44955 GHz; microwave power, 1.00 milliwatts; modulation
amplitude, 10.084 gauss; 15 K. PanelA is the high
field region of the spectrum of TMADH
at pH 10.0. PanelB is the half-field region of the spectrum of
TMADH
at pH 10.0. PanelC is the high
field region of the spectrum of TMADH
at pH 8.0. PanelD is the half-field region of the spectrum of
TMADH
at pH 8.0.
The visible absorbance spectrum of TMADH at pH 8 is intermediate between that seen at pH 6 and 10, showing
some of the features present in the pH 10 spectrum and suggesting that
two-electron reduced enzyme contains mostly flavin semiquinone and
reduced iron-sulfur center at pH 8 (Fig. 1, - - - -). However,
the features at 365 and 440 nm are not as well defined at pH 8 as they
are at pH 10, and the peak at 510 nm seen in the pH 10 spectrum is
virtually absent in the spectrum at pH 8, indicating that a significant
difference exists in TMADH
at these two pH values. These
differences appear to reflect changes in ionization of the flavin
semiquinone and, to a lesser degree, a shift in the electron
distribution toward further iron-sulfur reduction at the higher pH.
Differences between TMADH
at pH 8 and 10 is most
pronounced when examined by EPR. At pH 10, the EPR spectrum of
TMADH
reflects formation of the spin-interacting state (Fig. 2, A and B). At pH 8, however, the
high-field region of the EPR spectrum reflects a simple combination of
flavin semiquinone and reduced 4Fe/4S signals (Fig. 2C), and no half-field feature indicative of
spin-interaction is observed (Fig. 2D). EPR spectra of
samples at pH 8 were recorded at several microwave power levels in
order to facilitate quantitation of these signals (the flavin
semiquinone readily power saturates at the low temperatures required to
observe the reduced 4Fe/4S center). Spin integration of the axial
flavin semiquinone and rhombic 4Fe/4S EPR signals for TMADH
at pH 8 extrapolated to very low microwave power (<1
microwatts, so as not to saturate the flavin semiquinone signal)
confirms a 1:1 stoichiometry, i.e., an electron distribution
in which one reducing equivalent is on the FMN and one is on the
iron-sulfur center. This important result indicates that an
intramolecular electron distribution consisting of a flavin semiquinone
and a reduced iron-sulfur center is necessary, but not sufficient, to
produce the spin-interacting form of the enzyme.
One or more
ionizable groups with pK values > 8 must be
responsible for induction of the spin-interacting state in
TMADH
on raising the pH from 8 to 10. The pH dependence
of the g
4 signal intensity at 15 K is shown in Fig. 3A, and indicates that formation of the
spin-interacting state of the enzyme is controlled by a single
ionizable group exhibiting a pK
of 9.4. In an
effort to correlate formation of the spin-interacting state with
ionization of the neutral flavin semiquinone and to determine directly
the pK
for ionization of the semiquinone, the pH
dependence of the absorbance at 365 nm of TMADH
has been
examined. (
)The anionic form of flavin semiquinone displays
an absorbance band at this wavelength(19) , and the case of
trimethylamine dehydrogenase is relatively uncomplicated by the
spectral contribution of its iron-sulfur center. The pH dependence of
the 365 nm absorbance in TMADH
is shown in Fig. 3B, where it is evident that the absorbance grows
in over too great a pH range to be attributable to a single ionization.
A fit of the data using a two-pK
equation suggests
that full formation of the semiquinone anion requires the ionizations
of two groups exhibiting pK
values of 8.0 and 9.7,
the latter being within experimental error of that determined from the
pH dependence of the g
4 signal. One of these
ionizations must derive from the N-5 position of the flavin
semiquinone, while the other presumably derives from another site in
the protein, possibly hydrogen-bonded or otherwise associated with the
iron-sulfur center. The data indicate that three conditions must be met
for formation of the spin-interacting state in TMADH
: 1)
the distribution of reducing equivalents within the active site must
give flavin semiquinone and reduced iron-sulfur center; 2) the
semiquinone must be ionized; and 3) a third group within the active
site must also be deprotonated. All of these requirements are met at pH
10, but only the first (and possibly the second, to a greater or lesser
extent) is met at pH 8.
Figure 3:
pH dependence of EPR half-field signal
intensity and extinction coefficient at 365 nm. Panel A,
relative g = 4 signal intensity is plotted versus the pH at which the experiments were performed. The filledcircles represent the amount of g = 4
signal observed at a given pH, normalized to the amount seen at pH
10.0. The solidline represents a fit of the data to
a single pKexpression, yielding a value
of 9.4. PanelB, the maximum extinction coefficient
at 365 nm (filledcircles) during reductive titration
of TMADH with dithionite (at pH
8.0) or titanium citrate (at pH
< 8.0) is plotted versus pH. The filledcircles represent the extinction coefficients. The solidline represents a fit of the data to a double
pK
expression, yielding values of 8.0 and
9.7.
Figure 4:
Time
courses observed for the TMADH pH(D) jump reaction in
100% H
O and 100% D
O. Samples of two-electron
reduced trimethylamine dehydrogenase were prepared, and stopped-flow
rapid mixing experiments were performed as described under
``Material and Methods.'' The final enzyme concentrations
range from 50 to 100 µM. Absorbance changes observed at
365, 410, and 520 nm after mixing are plotted versus time. The solid lines represent fits of the data to exponential
expressions. The values of the observed rate constants for each
wavelength shown in the figure represent the average of at least six
independent measurements at each wavelength for a given set of
conditions. Panel A, pH 10
6 (100% H
O).
Data are fitted to a single exponential expression. Panel B,
pH 10
8 (100% H
O). Data are fitted to a single
exponential expression. Panel C, pD 10
6 (100%
D
O). Data are fitted to a single exponential expression. Panel D, pD 10
8 (100% D
O). Data collected
at time t
20 ms are fitted to a single exponential
expression in order to account for the lag phase. Panel E, pH
6
10 (100% H
O). Data are fitted to a single
exponential expression. Panel F, pH 8
10 (100%
H
O). Data are fitted to a single exponential expression. Panel G, pD 6
10 (100% D
O). Data are fitted
to a double exponential expression, and the fraction of absorbance
change attributed to each kinetic phase is given next to the value of
the observed rate constant. Panel H, pD 8
10 (100%
D
O). Data are fitted to a double exponential expression,
and the fraction of absorbance change attributed to each kinetic phase
is given next to the value of the observed rate constant. Panel
I, pH 8
6 (100% H
O). Data are fitted to a
single exponential expression. Panel J, pH 6
8 (100%
H
O). Data are fitted to a single exponential expression. Panel K, pD 8
6 (100% D
O). Data are fitted
to a single exponential expression. Panel L, pH 6
8
(100% D
O). Data are fitted to a single exponential
expression. Panel M, pD 8
6 (100% D
O) at
520 nm. The kinetic transient was collected at two time scales,
0-20 ms and 20-200 ms. Data are fitted to a
double-exponential
expression.
By contrast to the well behaved monophasic behavior
exhibited by the reactions described above, the 10 8 pD jump
reaction with TMADH
in D
O exhibits complex
kinetic behavior (Fig. 4D). The time courses show a
distinct lag phase at all three wavelengths monitored, followed by
monophasic kinetic behavior. Ignoring the lag phase, rate constants for
these time courses have been obtained by fitting the data points
collected at times
20 ms after mixing to a single exponential
expression. The results (Fig. 4D, solid lines)
give an observed rate constant for the 10
8 pD jump reaction of
60 s
. Comparison with the corresponding rate
constant in H
O gives an observed solvent kinetic isotope
effect of 7.0 for the pH(D) 10
8 reaction. The emergence of the
lag phase in the 10
8 pH(D) jump time courses as the mol
fraction of D
O present in the solvent increases (not shown)
suggests a multi-step mechanism for loss of the spin-interacting state
on jumping the pH(D) from 10 to 8.
pH jump experiments in the
reverse direction (i.e. pH(D) 6 10 and pH(D) 8
10) have also been performed. When performed in H
O, the
results are consistent with those obtained from the previous
trimethylamine dehydrogenase pH jump study(11) , with observed
time courses that exhibit fitted rate constants of 1000 s
for the 8
10 reaction and 930 s
for the
6
10 reaction. Again, well behaved monophasic time courses are
observed, with wavelength-independent rate constants (Fig. 4, E and F). In D
O, however, the observed
time courses are distinctly biphasic (Fig. 4, G and H), and a two-exponential expression is required to fit both
the 6
10 and 8
10 pD jump time courses satisfactorily (Fig. 4, G and H, solid lines). For
the 6
10 pD jump reaction, the rate constant for the fast
kinetic phase is 310 s
(which contributes
approximately half of the total observed absorbance change at all three
wavelengths monitored). The rate constant obtained for the slow kinetic
phase of the reaction, on the other hand, is wavelength-dependent,
ranging from 76 s
at 365 nm to 47 s
at 410 nm. This behavior suggests that the slow phase of the
reaction consists of multiple components having distinct spectral
changes, but which are kinetically unresolved. Multiphasic kinetics
makes the determination of an observed solvent kinetic isotope effect
for the pH(D) 6
10 jump problematic, but the effect is clearly
significant in the range of 3-15 (depending on whether rate
constants from the fast or slow phase of the reaction in D
O
are used in the calculation). As in the case for the D
O 10
8 pD jump experiment in D
O, the multiphasic behavior
exhibited by the 6
10 pD jump reaction suggests a complex
reaction mechanism.
The 8 10 pD jump reaction also gives
biphasic and wavelength-dependent time courses (Fig. 4H). At 365 and 520 nm, the fast phase of the
reaction gives a rate constant of 260 s
and accounts
for about half of the total absorbance change observed at these
wavelengths; the slow kinetic phase gives a rate constant of 46
s
. At 410 nm, the observed rate constant is 550
s
, significantly larger than those observed at 365
and 520 nm; the rate constant for the slow kinetic phase of the
reaction is 65 s
. Again, the implication is that the
spectral change associated with each of the two kinetic phases of the
reaction consist of multiple components. Calculating the solvent
kinetic isotope effect for the 8
10 pH(D) jump reaction is again
complicated by the multiphasic kinetics observed in D
O but
is in the range of 4-15.
For the 8 6 and 6
8 pH
jump experiments in H
O, the results are significantly
different than observed in the other pH jump experiments. In each of
the other cases (pH 8
10, 10
8, 6
10, and 10
6) the time courses are monophasic and observed rate constants
wavelength-independent for a given reaction. Wavelength-dependent rate
constants are observed only in the biphasic low-to-high pH(D) jump time
courses in D
O (Fig. 4, G and H).
In the case of the 8
6 pH jump in H
O, simple
monophasic behavior is observed, but the observed rate constants are
wavelength-dependent, ranging from 500 s
at
365-200 s
at 410 nm (Fig. 4I). (
)For the 6
8 pH jump reaction, time courses at 410
and 520 nm are monophasic with an observed rate constant of 400
s
(Fig. 4J). At 365 nm, however, no
kinetic absorbance change is observed, although an increase is expected
from the static difference spectrum (Fig. 1). It appears that
the spectral change at this wavelength occurs so rapidly that the
absorbance change is lost in the 600-µs dead time of the apparatus.
Again, the implication is that there are multiple components to the
overall spectral change associated with the reaction.
In all of the
pH(D) jump experiments performed up to this point, absorbance decreases
at 365 and 520 nm are observed along with an absorbance increase at 410
nm when the pH(D) is decreased and the reverse observed when the pH(D)
is increased (Fig. 4; (11) ). In both the 8 6 and
6
8 pD jump experiments in D
O, by contrast, the
direction of absorbance change at 365 nm is in the same direction as
that seen at 410 nm (Fig. 4, K and L) and
opposite to that observed at 365 nm in the corresponding H
O
experiment. At 520 nm, the transient exhibits rise-fall behavior (Fig. 4M). For the 8
6 pD jump reaction, the
365-nm kinetic transients collected at intermediate D
O mol
fractions exhibit rise-fall behavior with contributions from absorbance
changes in both directions (not shown). For the 6
8 pH(D) jump
reaction, a discernible absorbance change is observed at 365 nm only in
100% D
O, and is too small to permit a precise evaluation of
the observed rate constant (Fig. 4L).
A multi-step
kinetic scheme must be invoked to account for the above observations.
It appears that absorbance changes at 365 nm due to very rapid
reactions, which are lost in the dead time when performed in
HO, become slow enough to observe in D
O. As the
mol fraction of D
O increases, two distinct kinetic
processes are observed with absorbance changes in opposite directions
at both 365 and 520 nm. Given the nature of the experiments, the data
most likely reflect a protonation/deprotonation event (which is too
rapid to observe in H
O), followed by subsequent
intramolecular electron transfer.
A proton inventory
analysis for the pH jump experiments with TMADH is
straightforward only for the 10
6 pH(D) jump reaction, since
this is the only case where the time courses exhibit monophasic
kinetics and wavelength-independent observed rate constants at all
solvent mol fractions of D
O. In order to obtain rate
constants for the 10
8 pH(D) jump reaction, it is necessary to
ignore a plainly visible lag phase from the kinetic transients (Fig. 4D). In the case of the 8
6 and 6
8
pH(D) jump reactions, a proton inventory is possible if only the data
obtained at 410 and 520 nm are considered. Finally, the 6
10 and
8
10 pH(D) jump reactions display increasingly biphasic time
courses as the solvent mol fraction of D
O increases, and
neither the determination of an overall solvent kinetic isotope effect
nor a proton inventory analysis is justified for these reactions.
With these limitations in mind, a proton inventory analysis has been
performed for the 10 6, 10
8, 8
6, and 6
8
pH(D) jump reactions (Fig. 5). The proton inventory plot for the
8
6 pH(D) jump reaction is linear, consistent with the
involvement of only a single proton in the reaction (Fig. 5A, circles); the overall observed
solvent kinetic isotope effect is 1.7. The proton inventory plot for
the pH(D) jump in the reverse direction (6
8) is also linear,
with an observed overall solvent kinetic isotope effect of 3.2. While
the relative error in these stopped-flow rapid mixing experiments,
defined as the standard deviation of the mean of at least six
determinations, is too great to discriminate between one- and
two-proton mechanisms, it is clear that both reactions involve at least
one proton.
Figure 5:
Proton inventory analysis of TMADH pH(D) jump reactions. The observed rate constant divided by the
rate constant observed in 100% D
O is plotted versus the mol fraction of D
O present in the solvent. The error bars represent the standard deviation of the mean of at
least six independent measurements for a given set of conditions. Panel A, pH(D) 8
6 (filledcircles)
and pD 6
8 (filledtriangles). The solid
lines represent fits of the data to a linear expression. Panel
B, pH(D) 10
6 (filledsquares) and pD 10
8 (filleddiamonds). The solid lines represent fits of the data to the Gross-Butler equation (see (22) and references contained
therein).
For both the 10 6 and 10
8 pH(D) jump
reactions, the proton inventory plots are distinctly convex downward (Fig. 5B). In both cases, the precision of the kinetic
data are sufficient to conclude that a minimum of two protons are
involved(22) . This interpretation is consistent with the
static optical/EPR titration results (see above), which suggest that
there are at least two ionizable groups that exhibit pK
values > 8 and are important in controlling formation or
breakdown of the spin-interacting state. The precision of the kinetic
data is not high enough, however, to distinguish between two- and
three-proton reaction mechanisms. Taken together, the proton inventory
data indicate that there is at least one proton involved in the
reaction mechanism between pH(D) 6 and pH 8, and at least two protons
involved in the reaction mechanism between pH(D) 8 and 10. Thus there
must be at least three protons involved in the overall pH(D) jump
reaction mechanism between pH(D) 6 and 10.
The pH dependence of the static visible and EPR spectra from
this and previous studies indicate that three identifiable states of
TMADH are most easily observed at pH 6, 8, and 10. At pH
6, TMADH
appears to consist principally in the form
possessing flavin hydroquinone and oxidized iron-sulfur center. This is
consistent with the pK
value of 6 estimated for
the N-1 position of flavin hydroquinone from reductive half-reaction
studies(26) . At pH 8, TMADH
possesses mainly
flavin semiquinone and reduced iron-sulfur center, but the magnetic
moments of the unpaired spins do not interact at this pH. It is likely
that pH 8 is close enough to the pK
value for the
neutral/anionic flavin semiquinone equilibrium that significant amounts
of both forms are present. Finally, at pH 10, trimethylamine
dehydrogenase reduced by two equivalents possesses anionic flavin
semiquinone and reduced iron-sulfur center, with the magnetic moments
of the unpaired spins interacting strongly.
The data describe a
situation in which the relative reduction potentials of FMN and the
iron-sulfur center are such that an intramolecular electron
distribution consisting of flavin hydroquinone and oxidized iron-sulfur
is preferred at low pH, with the flavin hydroquinone predominantly
protonated at the N-1 position of the isoalloxazine ring. Recent work
on the reductive half-reaction of trimethylamine dehydrogenase with the
alternative substrate diethylmethylamine suggests that the N-1 position
of the flavin hydroquinone exhibits a pK value of
approximately 6 (26) . This value is comparable with the
pK
value for the N-1 position of free flavin
hydroquinone, which has been shown to exhibit a pK
value near 6.5(27) . This value is reasonably expected to
be lowered if the protein structure causes strain that introduces a
bend in the flavin about a line connecting atoms N-5 and N-10 of the
isoalloxazine ring, as is known to be the case for trimethylamine
dehydrogenase(28) . As the pH increases, loss of this proton to
produce flavin hydroquinone anion apparently decreases the reduction
potential for the hydroquinone/semiquinone couple below that for the
iron-sulfur center, so that by pH 8.0 the intramolecular electron
distribution favors flavin semiquinone and reduced iron-sulfur center
over the distribution consisting of anionic hydroquinone and oxidized
iron-sulfur by a factor of approximately four (26) . This
distribution is reflected in the EPR spectrum of trimethylamine
dehydrogenase reduced by two equivalents at pH 8, which appears to be a
simple combination of an axial flavin semiquinone radical signal and a
rhombic signal due to reduced iron-sulfur center (Fig. 2C). At pH 8, the FMN semiquinone appears to be
present mostly in the neutral form, as judged by the line width of its
EPR signal. A further increase in pH leads to ionization at the N-5
position of the flavin semiquinone (which exhibits a pK
of 8.0) to give the semiquinone anion. The semiquinone anion is
unable to become reduced by back electron transfer without uptake of a
proton (to do so would give rise to the very unstable dianionic flavin
hydroquinone), and at sufficiently high pH the distribution shifts
further in favor of iron-sulfur reduction over formation of the flavin
hydroquinone. The static optical/EPR titration and stopped-flow rapid
mixing kinetic data support the existence of still another ionizable
group within trimethylamine dehydrogenase, which exhibits a
pK
of approximately 9.5 (Fig. 3).
Deprotonation of this ionizable group as the pH is increased from 8 to
10 results in interaction of the magnetic moments of the unpaired spins
present on the flavin semiquinone anion and reduced iron-sulfur center.
Thus at sufficiently high pH, TMADH
is predominantly in
the form giving rise to the spin-interacting state, as evidenced by the
EPR signal exhibited by the enzyme (Fig. 2, A and B).
Low pH favors an electron distribution in TMADH in which the flavin is largely reduced and iron-sulfur center is
oxidized, whereas high pH favors formation of the flavin semiquinone
and reduced iron-sulfur center. This is consistent with the expected pH
dependence of the three reduction potentials of the system, with the
observed pH dependence of the intensity of the half-field EPR signal
(attributable to the spin-interacting state in which the iron-sulfur
center must be reduced), and with the intensity of the absorbance at
365 nm (attributable to the anionic flavin semiquinone) that is
observed with TMADH
. The semiquinone form of FMN can
exist in either the neutral (protonated) or anionic (deprotonated) form
depending on the ionization state of the N-5 position of the
isoalloxazine ring. Trimethylamine dehydrogenase has been shown to be
unusual among flavoproteins in that it can accommodate either of these
forms of flavin semiquinone depending on solvent pH, with a
pK
of approximately 8.0 (Fig. 3B; (11) ). Similarly, the hydroquinone of flavin can exist as
either the neutral or anionic form depending on the ionization state of
the N-1 position. The ionization of N-1 of the hydroquinone has been
inferred from the pH dependence of the reductive half-reaction of
trimethylamine dehydrogenase with diethylmethylamine(26) . The
reduction potentials of both the partially and fully reduced forms of
FMN increase with decreasing solvent pH since protonation of the
isoalloxazine ring neutralizes a negatively charged electron. In the
simplest formulation, the possible pH dependence of the iron-sulfur
center reduction potential has not been considered. The reduction
potential of the iron-sulfur center might also be expected to increase
with decreasing solvent pH for the same reason (e.g. protonation of a site near the 4Fe/4S cluster is expected to
increase the reduction potential). However, in determining the pH
dependence of TMADH
intramolecular electron distribution,
it is the relative reduction potentials of the two centers
that are important. Since the results show that low pH favors flavin
hydroquinone and oxidized iron-sulfur center, whereas high pH favors
flavin semiquinone and reduced iron-sulfur center, the flavin reduction
potential must increase relative to the iron-sulfur reduction potential
with decreasing solvent pH.
The kinetic results presented here,
particularly of the pD 6 8 and 8
6 experiments, indicate
that the reequilibration of reducing equivalents within TMADH
is a kinetically complicated process. We have previously reported
both static and kinetic difference spectra for the 10
7 and 7
10 pH jumps(11) . All of the spectral features observed
in the pH 10 minus pH 7 static difference spectrum for
TMADH
are quantitatively reproduced in the corresponding
kinetic difference spectra obtained from both the 10
7 and 7
10 pH jump reactions (although the two kinetic difference
spectra are necessarily opposite in sign). (
)Since the
static and kinetic difference spectra agree so well in these
experiments, we have concluded that there are no dead-time spectral
changes in the course of these reactions. This contrasts with the
results of the 6
8 pH jump experiments reported here,
particularly in the region around 365 nm, where there is a substantial
discrepancy between the kinetic spectral changes observed kinetically (Fig. 4) and those anticipated on the basis of the static
spectra at these two pH values (Fig. 1, dashed and dottedlines), presumably due to a significant dead
time spectral change in the kinetic experiments. The apparent
discrepancy between the 7
10 and 6
8 experiments is
resolved when one considers the difference in the state of
TMADH
at pH 6 versus pH 7; at pH 6 a substantial
portion of the flavin present as the hydroquinone is protonated,
whereas at pH 7 it exists predominantly as the anionic
FMNH
. If, during the low-to-high pH jump,
intramolecular electron transfer must be preceded by the ionization of
the N-1 position of flavin hydroquinone and this ionization exhibits a
pK
of 6 (both of these assertions are supported by
recent studies of the reductive half-reaction; (26) ), then
approximately 90% of TMADH
at pH 7 already exists in the
form (anionic flavin hydroquinone and oxidized iron-sulfur center)
observed at the conclusion of the 6
8 pH jump reaction. In other
words, the absorbance increase at 365 nm, which is lost in the dead
time of the mixing apparatus in the 6
8 pH jump, is undetectable
as a dead time spectral change in the 7
10 pH jump reaction
since only 10% of the enzyme molecules undergo this process.
The
multiphasic kinetic behavior and observation wavelength dependent rate
constants observed in the 10 8, 8
10, and 6
10
100% D
O pD jump time courses coupled with the mol fraction
D
O-dependent direction of absorbance change observed in the
8
6 and 6
8 100% D
O pD jump time courses
eliminate the possibility that the protonation/deprotonation and
intramolecular electron transfer events occur concomitantly and
indicate instead a reaction mechanism that entails discrete
protonation/deprotonation and electron transfer steps. Fig. S1represents the simplest overall reaction
mechanism for prototropically controlled electron transfer within
TMADH
that is consistent with the known kinetic behavior
of the enzyme.
Figure S1: Scheme 1
The mechanism consists of discrete equilibria involving three prototropic equilibria and an intramolecular electron transfer step. Two of the three prototropic equilibria shown in Fig. S1involve ionizations of the flavin N-1 of hydroquinone and position N-5 of the semiquinone. The third prototropic equilibrium is between the protonated and unprotonated forms of an as yet unidentified ionizable group whose ionization is required for formation of the spin-interacting state. The intramolecular electronic equilibrium involves electron transfer from anionic flavin hydroquinone to oxidized iron-sulfur center (to give neutral flavin semiquinone and reduced iron sulfur center) and the reverse reaction.
In the course of a 10
6 pH jump experiment, the unknown group and the N-5 position of
anionic flavin semiquinone are first protonated, disrupting the
interaction of the unpaired spins and forming the neutral flavin
semiquinone. The order of protonation might be the reverse of that
shown in Fig. S1, or protonation of these sites may occur
simultaneously. Initial protonation of the unknown group followed by
protonation of the anionic flavin semiquinone as drawn, however, is
consistent with the observation of a lag phase in the 100%
D
O 10
8 time courses, and also with the results of
reductive half-reaction studies with the slow substrate
diethylmethylamine, which indicate that formation of the
spin-interacting state is kinetically distinct from intramolecular
electron transfer, with little or no absorbance change associated with
it(26) . (
)Subsequent to these two protonations,
electron transfer from the iron-sulfur center to FMNH
forms the
anionic hydroquinone, which finally protonates (at sufficiently low pH)
to give the neutral hydroquinone.
In principle, all of the active
sites in a sample of TMADH will possess the neutral
flavin hydroquinone at sufficiently low pH. At pH 6 (the lowest pH used
in this and previous work and approximately the pK
of the hydroquinone) approximately 50% of the enzyme possesses
the neutral form of the flavin hydroquinone(26) . The remaining
50% of the active sites should consist of a mixture of (anionic flavin
hydroquinone with oxidized iron-sulfur center), and (neutral flavin
semiquinone and reduced iron-sulfur center) in a ratio of
4:1. The
visible absorbance spectrum of the enzyme species at the far left of Fig. S1has been independently determined from reductive
half-reaction studies of trimethylamine dehydrogenase since initial
two-electron reduction of the FMN by substrate can be kinetically
resolved from other processes taking place(7, 26) ;
this is shown in Fig. 1(opencircles).
Given that a simple two-exponential expression provides a
satisfactory fit to the vast majority of the kinetic data presented
here, kinetic simulations based on a mechanism with as many floating
variables (eight rate constants) as depicted by Fig. S1would
certainly lead to meaningless fits to the data, and these were not
attempted. As an alternative means to assess whether the proposed
scheme is consistent with the data, an attempt has been made to account
for the pH dependence of the observed rate constants for electron
transfer within TMADH in terms of Fig. S1. It is
known that the observed rate constant for electron transfer within
TMADH
in H
O depends only on the final pH of
the experiment(11) . As shown in Fig. 6, a plot of k
versus final pH plot is U-shaped with
a minimum near pH = 7.5, a reflection of the complex reaction
mechanism required to describe prototropic control of intramolecular
electron transfer in this enzyme. Since ionization of the unknown group
with pK
9.5 perturbs the electron distribution within
partially reduced trimethylamine dehydrogenase only very slightly (as
reflected in the EPR spectra observed at pH 8 and 10), consideration of
only the FMN hydroquinone and semiquinone ionization states is to a
good approximation sufficient to account for the observed pH dependence
of k
. Omitting the far right equilibrium of Fig. S1gives Fig. S2, in which the several microscopic
rate constants for the interconversion of all intermediates are given
explicitly.
Figure 6:
pH
dependence of the observed rate constant for the TMADH pH
jump. The average of pH jump (100% H
O) k
values obtained at 25 °C (taken from this work and (11) ) plotted versus the experimental final pH
(
). The solid line represents a fit of the k
versus pH data to using
pK
values of 6.0 and 8.5 for the FMN
hydroquinone and semiquinone respectively and rate constants for the
electron transfer reactions (k
and k
) = 1000 s
(see text).
The fitted values for the bimolecular rate constants for protonation of
the FMN hydroquinone and semiquinone are 2.6
10
M
s
and 1.0
10
M
s
, respectively.
Figure S2: Scheme 2
Since the pH remains constant subsequent to the pH jump, the time
rate of change for [[H] is zero, and
each of the protonation steps can be considered pseudo first-order. Fig. S2is of the form A
B
C
D. In situations in which the appearance of final product (either
A or D depending on the direction of the pH jump) follows first-order
kinetics, as is observed in the present pH jump reactions in
H
O, an expression can be derived for the observed
first-order rate constant (k
) as a function of
pH and the microscopic rate constants that describe the three
equilibria (making a steady state approximation for the rate of change
of intermediates B and C; i.e.dB/dt = dC/dt = 0). Making these
assumptions, the observed rate constant for intramolecular electron
transfer as a function of proton concentration is given by the
following equation. (
)
The rate constants for the protonation of the flavin
hydroquinone (k) and semiquinone (k
) anions are equal to the corresponding
bimolecular association rate constants multiplied by the proton
concentration (i.e.k
= k`
and k
= k`
[H
]). The rate constants for deprotonation (k
and k
) are then defined in
terms of the corresponding pseudo first-order protonation rate
constants, the pK
values of the appropriate flavin
species, and the final pH value used in the experiment (i.e.k
= k
(10
) with
= pH
-
pK
and k
= k
(10
) with
=
pH
- pK
). The rate
constants for electron transfer (k
and k
) are considered pH-independent parameters, reflecting the intrinsic rates of electron transfer
from the reduced iron-sulfur center to the neutral semiquinone and from
the anionic hydroquinone to the oxidized iron-sulfur center,
respectively.
In fitting the kversus final pH data to the above equation (Fig. 6, solid
line), pK
values of 6.0 for the flavin
hydroquinone and 8.5 for the semiquinone were used. The former value is
consistent with recent reductive half-reaction studies of
trimethylamine dehydrogenase(26) . In the case of the
pK
for the semiquinone, several attempts were made
to fit the data using different values for the semiquinone
pK
, and it was found that a value of 8.5 yielded
the best result. This value is within the range established by the
optical/EPR spectral data (11) and the present kinetic results.
The lower limit for the rate constants associated with the
intramolecular electron transfer steps (k
and k
) is 1000 s
; fits of the k
versus pH data to at
different fixed values of k
and k
below 1000 s
were unsatisfactory (data not
shown). From the parameters giving the best fit to the pH dependence of k
, the bimolecular rate constants for
protonation of the hydroquinone and semiquinone are calculated to be
2.6
10
M
s
and 1.0
10
M
s
, respectively.
When considering the
interrelationship between prototropic equilibria and intramolecular
electron transfer in trimethylamine dehydrogenase, the
protonation/deprotonation and electron transfer events may either occur
concomitantly or as discrete chemical steps. Evidence for a discrete
mechanism has been found in the kinetic and thermodynamic behavior of
medium chain acyl-CoA dehydrogenase(29) , while clear evidence
for a concomitant mechanism has been found in the case of electron
transfer within xanthine oxidase(30) . The present results
indicate that electron transfer within trimethylamine dehydrogenase
operates via a mechanism involving discrete ionization and electron
transfer steps. The kinetic transients under many reaction conditions
are complex (with either lags or multiple phases); the proton
inventories indicate multiple ionizations involved in a pH 6 10
jump or the reverse and the pH dependence of the reaction in
H
O are distinctly nonlinear. Xanthine oxidase, by contrast,
exhibits well behaved kinetics under all conditions, linear proton
inventory plots indicative of the involvement of only a single proton
in the electron transfer process, and a linear dependence of k
on pH. These results are not contradictory but
are simply a reflection of different systems operating by different
mechanisms.
Two factors in all likelihood combine to determine which type of mechanism is found in a given system. The first of these is the flavin redox couple participating in electron transfer; this is the quinone/semiquinone couple in xanthine oxidase and the semiquinone/hydroquinone couple in trimethylamine dehydrogenase. Since both semiquinone and hydroquinone, but not oxidized quinone, oxidation states of the flavin have ionizable protons, there is clearly greater likelihood for participation of multiple protons in the latter case than in the former. The second factor is the pK of the reduced form of the flavin that participates in the reaction. Thorpe and co-workers (29) point out that during the oxidation of a neutral semiquinone or hydroquinone, deprotonation to form the corresponding anionic species must precede electron transfer so as to avoid formation of an unfavorable cationic flavin species. Similarly, protonation of an anionic semiquinone should occur prior to reduction in order to avoid formation of an unfavorable dianionic hydroquinone. The protonation state of the flavin thus controls the kinetics as well as the thermodynamics of intramolecular electron transfer. For these reasons, a discrete mechanism for coupled proton/electron transfer might be expected for a given system in the absence of other considerations, as is observed in the cases of acyl-CoA dehydrogenase and trimethylamine dehydrogenase. However, should the polypeptide preferentially destabilize the deprotonated form of the flavin by virtue of its hydrogen bonding and other interactions with the isoalloxazine ring, then a discrete pathway might not necessarily reflect the lowest energy path from the initial to the final states in the electron transfer process, in which case concomitant electron/proton transfer is preferred. This is apparently the case with xanthine oxidase.
The present results extend previous work indicating that electron transfer from the flavin hydroquinone of trimethylamine dehydrogenase to the iron-sulfur center of the enzyme is quite fast and is not intrinsically rate-limiting in catalysis. Furthermore, evidence is found that formation of the spin-interacting state observed at the completion of the reaction of oxidized enzyme with substrate is governed by an ionizable group having a pK of approximately 9.5. Ionization of the flavin semiquinone to its anionic form also occurs in the course of formation of the spin-interacting state, with a pK of approximately 8.0. Given that only two redox-active centers are present in trimethylamine dehydrogenase, the enzyme might be considered a relatively simple system in which to examine electron transfer. Despite this apparent simplicity, the present results indicate a rather complicated reaction mechanism for the internal equilibration of reducing equivalents between the two centers that involves at least three discrete ionizations. These results combined with those obtained from other studies further indicate that two of these three ionizable groups are associated with the FMN coenzyme (in the semiquinone and hydroquinone oxidation state, respectively), while the third is most likely associated with an amino acid residue located at or near the active site(11, 26) .