From the Department of Biochemistry, University of Mississippi
Medical Center, Jackson, Mississippi 39216-4505
The pH dependence of the redox potentials for the
oxidized/reduced couples of methylamine dehydrogenase (MADH) and
aromatic amine dehydrogenase (AADH) were determined. For each enzyme, a change of
30 mV/pH unit was observed, indicating that the
two-electron transfer is linked to the transfer of a single proton.
This result differs from what was obtained from redox studies of a
tryptophan tryptophylquinone (TTQ) model compound for which the
two-electron couple is linked to the transfer of two protons. This
result also distinguishes the redox properties of the enzyme-bound TTQ
from those of the membrane-bound quinone components of respiratory and
photosynthetic electron transfer chains that transfer two protons per
two electrons. This difference is attributed to the accessibility of
TTQ to solvent in the enzymes. One of the quinol hydroxyls is shielded
from solvent and thus is not protonated. The unusual property of TTQ
enzymes of stabilizing the anionic form of the reduced quinol is
important for the reaction mechanism of MADH because it allows
stabilization of physiologically important reaction intermediates.
Examination of the extent to which disproportionation of the MADH and
AADH semiquinones occurred as a function of pH revealed that the
equilibrium concentration of semiquinone increased with pH. This
indicates that the proton transfer is linked to the semiquinone/quinol
couple. Therefore, the quinol is singly protonated, and the semiquinone
is unprotonated and anionic. It was also shown that the
oxidation-reduction midpoint potential for AADH is 20 mV less positive
than that of MADH over the range of pH values that was studied and that
the TTQ semiquinone of AADH was less stable than that of MADH. This may
be explained by differences in the active site environments of the two
enzymes, which modulate their respective redox properties.
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INTRODUCTION |
Redox reactions involving proteins are ubiquitous processes that
are fundamental to respiration, photosynthesis, and reactions of
intermediary metabolism. Enzyme-bound quinones and flavins are
important cofactors of oxidoreductases that function in biological catalysis and electron transfer. These prosthetic groups, as well as
membrane-bound components of respiratory electron transfer chains, are
particularly important because they are able to couple the two-electron
oxidation of substrates to single electron carriers during metabolism
and respiration.
Methylamine dehydrogenase (MADH; reviewed in Ref. 1)1
catalyzes the oxidation of methylamine in the periplasm of many
methylotrophic and autotrophic bacteria to form ammonia and
formaldehyde concomitant with the two-electron reduction of its redox
cofactor (Reaction 1). MADH is
an H2L2 heterotetramer with subunit
molecular masses of 47 and 15 kDa. The tryptophan tryptophylquinone
(TTQ; Fig. 1) (2) prosthetic group, which
is located on each small subunit, is derived from two tryptophan side
chains that are covalently cross-linked and contain an orthoquinone
function through a post-translational modification. In autotrophic
bacteria, such as Paracoccus denitrificans, the
substrate-derived electrons are subsequently passed to a type I copper
protein, amicyanin (3), then to one or more c-type cytochromes (4), and finally to a membrane-bound cytochrome oxidase.
The crystal structure of a ternary complex of MADH, amicyanin, and
cytochrome c-551i from P. denitrificans has been
determined (5). The second TTQ-dependent enzyme to be
characterized thus far is aromatic amine dehydrogenase (AADH) from
Alcaligenes faecalis (6, 7). It catalyzes the same reaction
shown as Reaction 1, except that its preferred substrate is not
methylamine but rather phenylethylamines. Like MADH, AADH uses a type 1 copper protein, in this case azurin, as an electron acceptor (8, 9). These enzymes are members of a newly characterized class of primarily soluble enzymes, referred to as quinoproteins or quinoenzymes (1, 10,
11), which utilize covalently or tightly bound quinones as prosthetic
groups.

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Fig. 1.
The structure of oxidized tryptophan
tryptophylquinone (TTQ). The C-6 and C-7 positions are
labeled.
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That TTQ-dependent enzymes donate electrons to another
protein that is a one-electron carrier is of interest for two reasons. (i) This requires that the oxidative half-reaction proceeds via a
semiquinone intermediate. This means that electron transfer from TTQ to
copper occurs from two different redox states of TTQ during the
catalytic cycle. (ii) The electron acceptors for most oxidoreductases
use small molecules such as NAD+ or O2. When
these electron acceptors are present at the active site of the enzyme,
it is possible to directly transfer protons, as well as electrons, from
substrate or cofactor to acceptor. This cannot occur in these
quinoproteins. Their electron acceptors are other proteins that cannot
diffuse into the enzyme active site. The electron acceptors for MADH
and AADH are reduced by long range electron transfer. Thus, protons and
electrons cannot be transferred together. This raises questions as to
the protonation states of the reduced and semiquinone forms of the TTQ
cofactor during the catalytic cycle and the fate of the
substrate-derived protons. It also suggests functional similarities
with membrane-bound quinones in the respiratory chain, such as
ubiquinone, which must also separate electron and proton transfer as a
part of their physiologic redox reaction (12, 13).
Although much is known about the structure and reactivity of MADH,
relatively little is known about its redox properties. The
oxidation-reduction midpoint potential (Em)
value for the two-electron oxidized/reduced redox couple of P. denitrificans MADH was determined to be +100 mV at pH 7.5 (14). A
similar value was reported for MADH from bacterium W3A1 (15). Recently, the redox properties of a TTQ model compound were reported (16). It
exhibited an Em value similar to that of MADH at
pH 7.5. The Em value of the model compound was
pH-dependent, and it was shown that two protons were
transferred per two electrons during the conversion from fully oxidized
to fully reduced.
In this paper, we report for the first time the pH dependence of
the redox potential for a quinoprotein. The Em
values for the oxidized/reduced couples of MADH and AADH were each
determined over a range of pH values, and the Em
value of each enzyme exhibited a pH dependence different than that of
the TTQ model compound and different from that which is typically
observed for biologically active free and membrane-associated quinones.
The relative dependence on pH of the one-electron oxidized/semiquinone
and semiquinone/reduced couples of MADH and AADH was determined by
examining the extent to which disproportionation of each semiquinone
occurred as a function of pH. This allowed determination of the
protonation states of the TTQ quinol and semiquinone in MADH and AADH.
Differences in the redox properties of the protein-bound TTQ and the
TTQ model compound and other biogenic quinones are explained by
correlation with the crystal structure of MADH (17, 18). The relevance of these findings to the reaction mechanisms of MADH and AADH is
discussed. Comparison of the redox properties of MADH and AADH has also
allowed us to infer that differences in the active site structures of
the two enzymes further modulates their respective redox
properties.
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EXPERIMENTAL PROCEDURES |
Amicyanin (3) and MADH (19) were purified from P. denitrificans (ATCC 13543) as described previously. MADH and
amicyanin concentrations were calculated from known extinction
coefficients (3, 14) in 10 mM phosphate buffer, pH 7.5. The
extinction coefficients of the different redox forms of MADH vary with
pH (20) and ionic composition of the buffer (21). Purifications of AADH
(7) and azurin (8, 22) from A. faecalis (IFO 14479) were as
described previously, and protein concentrations were calculated from
previously determined extinction coefficients (7, 23). All reagents
were obtained from Sigma. Absorption spectra were recorded with a
Milton Roy Spectronic 3000 Array spectrophotometer.
Redox titrations were performed at 25 °C in either 10 mM
potassium phosphate buffer or 10 mM BisTris-propane buffer.
All Em values are reported relative to the
standard hydrogen electrode. Control experiments indicated that the
choice of buffer did not affect the experimentally determined
Em values. During redox titrations of MADH and
AADH, phenazine methosulfate (PMS) was used to monitor the ambient
redox potential. PMS is an ideal choice, because its Em value is similar to that of MADH and AADH and
because the absorption spectra of the oxidized and reduced forms of PMS
are distinct and exhibit minimal overlap with the spectra of oxidized
and reduced MADH and AADH (Fig.
2A). Given these absolute
spectra, it was possible to deconvolute the spectra of mixtures of
different redox forms of PMS and either MADH or AADH and to determine
the concentration of each species (discussed under "Results").
Ratios of MADHox/MADHred and
AADHox/AADHred were calculated so that the
Em value could be determined from plots of
E versus log(ox/red) at each pH. The ambient
potential was calculated from the ratios of
PMSox/PMSred. The Em
value for PMS is +80 mV at pH 7.0 and 25 °C, and it varies by 29.7 mV per pH unit over the range of pH studied, since the redox reaction
involves the transfer of one proton per two electrons (24). The redox
properties of PMS were verified by differential pulse voltammetry using
a Cypress Systems electroanalytical system. PMS was stable over the
range pH 6.0-8.5, which covers the physiologic pH range for these
enzymes. In addition to PMS, phenazine ethosulfate (Em = +60 mV at pH 7.0) and 1,2-naphthaquinone
(Em = +140 mV at pH 7.0) were also used as
mediators. Under identical reaction conditions, titrations using the
other mediators yielded results very similar to those obtained with
PMS. PMS was the mediator of choice because it exhibited better
spectral resolution and faster equilibration times than the other
mediators over the range of pH examined.

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Fig. 2.
A, absorption spectra of PMS
and MADH. All spectra were recorded in 10 mM potassium
phosphate, pH 6.8, at 25 °C. a, fully oxidized spectrum
of 20 µM MADH; b, spectrum of 20 µM MADH after reduction by methylamine; c,
fully oxidized spectrum of 33 µM PMS; d, fully
reduced spectrum of 33 µM PMS. B, oxidative
titration of reduced MADH. The sample mixture contained 10 mM MADH and 16.5 µM PMS in 10 mM
potassium phosphate, pH 6.8, at 25 °C. The first spectrum is that of
the mixture that contains fully reduced MADH. The oxidative titration
was performed as described under "Experimental Procedures." The
arrows indicate the directions of the spectral
changes.
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Reductive titrations were performed anaerobically. Anaerobic
conditions were maintained as described previously (25). During reductive titrations, either ascorbic acid or the substrate
(methylamine for MADH and tyramine for AADH) was added incrementally.
There is no direct reaction between either methylamine or tyramine and PMS, while methylamine will completely reduce PMS through MADH in less
than 3 s. Oxidative titrations exploited the fact that mixtures of
PMS with MADH or AADH are slowly oxidized by O2 in the dark
under aerobic conditions. Air was introduced to the fully reduced
sample, and spectra were recorded with time. Titration was performed in
the dark to avoid light-induced side reactions of PMS (24) or enzyme
(26). The reactions were complete in 30 min for AADH and 3 h for
MADH. Em values were determined from titrations
in both the reductive and oxidative directions, and essentially
identical results were obtained.
The semiquinone forms of MADH and AADH were generated by the
stoichiometric addition of sodium dithionite (7, 14). These semiquinones are kinetically stabilized. With time, each semiquinone disproportionates to a mixture of oxidized and reduced enzyme. The
extent to which disproportionation occurs depends on pH. The semiquinone samples were incubated for 12 h in 50 mM
BTP buffer at pH 5.0, 7.0, 9.0, and 10.0. Spectra of each were recorded
with time until the spectral changes had stabilized (i.e.
the system had reached equilibrium). From the known spectrum of each
redox form, the spectrum of each mixture was deconvoluted to estimate the equilibrium concentration of each redox form.
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RESULTS |
Redox Titrations of MADH--
PMS was used to monitor the ambient
redox potential during redox titrations of MADH. The spectra of the
fully oxidized and reduced forms of MADH and PMS are shown in Fig.
2A. MADH and PMS each exhibit an isosbestic point at
approximately 361 nm. This is clearly seen during the redox titration
of MADH (Fig. 2B), and it provides a convenient reference
point. To calculate the exact ratio of concentrations of the oxidized
and reduced forms of each species, data files containing the recorded
absorbance at each wavelength were analyzed. No semiquinone form of
either MADH or PMS was observed during these titrations, so
contributions to the spectra from those redox forms were not
considered. Each recorded spectrum is viewed as a matrix that is a
linear combination of the spectra of MADHox,
MADHred, PMSred, and PMSox. These
data sets were fit to Equation 1,
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(Eq. 1)
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where k1/k2 is
the ratio of PMSox/PMSred, and
k3/k4 is the ratio of
MADHox/MADHred. E was then
calculated using Equation 2.
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(Eq. 2)
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For PMS, the Em value is +80 mV at pH 7.0 and 25 °C, and 2.3RT/nF = 29.7 mV.
Over the range of pH values studied, the Em of
PMS will vary by 29.7 mV/pH unit, since the redox reaction involves the
transfer of one proton per two electrons (24). To calculate the
Em value of MADH at any pH,
log(k3/k4) was plotted
against E (Fig. 3), and data
were fit to Equation 3. Titrations were completely reversible, and the
same Em values were obtained from the reductive
and oxidative titrations (Fig. 3).
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(Eq. 3)
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The Em value of MADH at pH 7.5 was
determined to be 95 ± 5 mV. This is equivalent to the value of
100 ± 4 mV, which was determined by spectroelectrochemical
titration of MADH at this pH (14). Titrations using phenazine
ethosulfate and 1,2-naphthaquinone yielded the same
Em value as that obtained with PMS.

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Fig. 3.
Redox titration of MADH. Titrations were
performed in 10 mM potassium phosphate, pH 6.8, at
25 °C. In this titration, methylamine is used as a reductant, and
O2 is the oxidant. Data points include those obtained
during both the reductive titration ( ) and the oxidative titrations
( ).
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In reductive titrations, essentially identical results were obtained
when ascorbic acid or methylamine was used as a reductant. Reduction by
ascorbic acid will yield a quinol form of MADH with oxygen bound to the
C-6 of TTQ (O-quinol). Reduction by methylamine will yield
an aminoquinol form of MADH with a substrate-derived amino group bound
to C-6 of TTQ (N-quinol) (27). The identical Em values obtained from reductive titrations
using ascorbate and methylamine may be explained by the fact that these
are equilibrium measurements. When N-quinol is oxidized to
the quinone, the amino group is lost. Consequently, the
oxidation-reduction of the N-quinol is not a reversible reaction.
During the redox titrations, after equilibration the initially formed
N-quinol will probably be converted to O-quinol.
Therefore, the Em values reported in this study
are that of the quinone/O-quinol couple, regardless of which
reductant is used.
Redox Titrations of AADH--
Redox titrations of AADH were
performed exactly as described above for MADH. The results of these
studies were very similar with two notable differences. The
equilibration time for AADH during the oxidative titration was
approximately half of that for MADH. Also, the
Em value of AADH at any given pH was less positive than that of MADH (see below).
pH Dependence of the Em Values of MADH and
AADH--
The Em values of MADH were determined
over a range of pH. The plot of Em
versus pH (Fig. 4) is linear
over the range of pH examined and exhibits a slope
30.1 ± 1.5 mV per pH unit. This indicates that the two-electron redox reaction is
linked to the transfer of one proton (24, 28). Since the oxidized
quinone is not protonated, this means that only one of the hydroxyl
groups on the fully reduced quinol TTQ in MADH is protonated.

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Fig. 4.
Dependence on pH of the
Em values of MADH and AADH. For titrations
of MADH, either methylamine ( ) or ascorbate ( ) was used as a
reductant. For titrations of AADH ( ) tyramine was used as a
reductant.
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Since the plot in Fig. 4 is essentially linear, with no inflection
points on either end, it is not possible from these data to determine a
pKa value for either the quinone or quinol. As with
a ketone, the pKa for the quinone oxygens is expected to be very low, and it is not surprising that the acidic end
of the curve remains linear. The oxidized cofactor is, therefore, unprotonated TTQ. For the reduced form, one may conclude from these
data that the pKa value for the hydroxyl group that
is protonated is greater than 8.5. The reduced cofactor is TTQH
. A pKa > 8.5 is expected for
organic O-quinols and consistent with the
pKa for the TTQ model compound of 10.1 (16). In the
model compound, however, both hydroxyls are protonated. It was not
possible to obtain pKa values for MADH, because exposure to nonphysiologic pH values outside the range used in this
study causes denaturation or nonspecific structural perturbations of
the enzyme.
The Em values of AADH were determined at
different values of pH (Fig. 4). The plot of Em
versus pH for AADH is also linear and exhibits a slope that
is essentially the same as that for MADH. Thus, for AADH as well, only
one of the quinol hydroxyl groups is protonated in the fully reduced
enzyme. The Em values for AADH were less
positive than those for MADH by approximately 20 mV at each pH
value.
pH Dependence of the Disproportionation Reactions of the MADH and
AADH Semiquinones--
Although no semiquinone was observed during the
redox titrations, semiquinone forms of these enzymes can be generated
in vitro and are of physiologic importance in
vivo. Since the two-electron interconversion of quinone and quinol
involves the transfer of only one proton, this raises the question of
whether it is the quinone/semiquinone or semiquinone/quinol couple that
is linked to the proton transfer. It was not possible to measure the
Em values for the individual one-electron
couples in this study. At physiologic pH, the semiquinone is
kinetically, not thermodynamically, stabilized. In the presence of
mediators it is unstable, because they facilitate the transfer of
electrons between cofactors and enhance the rate of disproportionation.
It was possible, however, to determine which of the one-electron
couples is linked to proton transfer by examining the pH dependence of
the extent of the disproportionation reaction of the semiquinone.
If proton transfer is linked to the interconversion of quinol and
semiquinone (Reactions 2), then the Em value
for the quinone/semiquinone couple will be independent of pH, and the
Em value for the semiquinone/quinol will vary
with pH by approximately
60 mV/pH unit.
If this were true, then for the disproportionation of the
semiquinone (Reaction 4) the equilibrium concentration of TTQ·
would increase with pH. The disproportionation reaction will proceed to
a lesser extent at high pH than at low pH. If proton transfer is linked
to the interconversion of semiquinone and quinone (Reactions 5 and 6),
then the Em value for the quinone/semiquinone couple will vary with pH by approximately
60 mV/pH unit, and the
Em value for the semiquinone/quinol will be
independent of pH.
If this were true, then for the disproportionation of the
semiquinone (Reaction 7) the equilibrium concentration of TTQH·
would decrease with pH. The disproportionation reaction will proceed to
a lesser extent at low pH than at high pH.
Because the semiquinone forms are kinetically stabilized, it is
possible to generate a relatively stable semiquinone by titration with
dithionite. With time, however, the semiquinone will disproportionate to the reduced and oxidized form until an equilibrium is established (Fig. 5A). As discussed
earlier, the semiquinone is not stable during redox titrations, because
the presence of mediators significantly enhances the rate of
disproportionation. The semiquinones of MADH and AADH were generated,
and the composition of the final equilibrium mixture was monitored
after incubation in buffers at pH 5, 7, 9, and 10 (Fig. 5B).
Essentially identical results were obtained for MADH and AADH. The only
difference was that the rate of the disproportionation reaction of AADH
was faster than that of MADH. The extinction coefficients for the
different redox states of MADH and AADH vary with pH in the presence of
monovalent cations (20, 21). For this reason, BTP buffer was used. For
each enzyme, the most semiquinone is observed in the equilibrium
mixture at pH 10.0, and somewhat less is observed at pH 9.0. At pH 7.0 and 5.0 the semiquinone has completely disproportionated into a mixture of reduced and oxidized enzyme. Given the known spectrum of each redox
form, it was possible to deconvolute the spectra in Fig. 5B
and then determine the concentrations of each redox form present in the
mixture after equilibrium had been established. The pH dependence of
the proportion of MADH semiquinone present at equilibrium is seen in
Fig. 6A. The equilibrium
concentration of semiquinone increases at high pH. The solid
line in Fig. 6A is derived from Reactions 2 and describes the predicted fraction of semiquinone if proton transfer
is linked to interconversion of the quinol and semiquinone. The
dashed line is derived from Reactions 5 and describes the predicted fraction of semiquinone if proton transfer is
linked to interconversion of the semiquinone and quinone. From these
data, it is evident that proton transfer is linked to the interconversion of the fully reduced quinol and semiquinone forms (Reactions 2). The semiquinone form must, therefore, be the
unprotonated anionic semiquinone.

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Fig. 5.
A, disproportionation of AADH. The
semiquinone of AADH (5 µM) was incubated anaerobically in
0.05 M BTP, pH 7.0. Spectra were recorded at 0, 165, 504, 894, 1588, and 2641 s. The arrows indicate the
direction of the spectral changes. B, pH dependence of the
disproportionation reaction of MADH. The semiquinone of MADH (5 µM) was incubated anaerobically for 12 h, in 0.05 M BTP buffer at pH 5.0 (a), pH 7.0 (b), pH 9.0 (c), and pH 10.0 (d). The
spectra that are displayed were recorded after all spectral changes had
ceased and the system was at equilibrium.
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Fig. 6.
A, pH dependence of the
disproportionation reaction of MADH. The fraction of MADH
present as semiquinone after the disproportionation reaction had
reached equilibrium was calculated by deconvoluting the spectra shown
in Fig. 5B using the known spectra of the oxidized,
semiquinone, and reduced forms. The solid line is
the fraction of semiquinone predicted to be present according to the
model described by Reactions 2. The dashed
line is the fraction of semiquinone predicted to be present
according to the model described by Reactions 5. Each
line assumes a pK value of 10 for the reduced
MADH quinol. B, predicted pH dependence of the
Em values for the two one-electron couples of
MADH. Dashed line, semiquinone/reduced
one-electron couple; dotted line,
oxidized/semiquinone one-electron couple; solid
line, oxidized/reduced two-electron couple.
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The redox potentials for the two one-electron couples of MADH were
previously determined (29) by kinetic techniques at pH 7.5 at 10 °C
to be +190 mV (semiquinone/reduced) and +14 mV (oxidized/semiquinone), which corresponds to an Em value of +102 mV for
the two-electron couple. After correction for temperature differences,
this value is within experimental error of the value determined at pH
7.5 in the present study. The results presented above indicate that for
MADH the Em value of the oxidized/semiquinone
couple will not vary with pH, and that of the semiquinone/reduced
couple will vary by approximately 60 mV/pH unit (i.e. one
proton transfer is linked to one electron transfer). Thus, while it was
not possible to directly measure the Em value
for each one-electron couple, the results obtained here do allow one to
estimate these redox potentials over the range of pH studied (Fig.
6B).
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DISCUSSION |
These results reveal the protonation states of the fully reduced
and semiquinone forms of TTQ in MADH and AADH. The quinol is singly
protonated, and the anionic semiquinone is unprotonated. It was also
shown that the Em value for AADH is 20 mV less
positive than that of MADH. An important aim of this study is to
determine how the protein influences the redox properties of the TTQ
prosthetic group and whether this influences the catalytic properties
of the protein-bound quinone in the quinoprotein. Itoh et
al. (16) performed a detailed analysis of the redox properties of
a model compound of TTQ in which methyl groups are present at the
positions where the rings are covalently attached to the protein.
Differences in the redox properties of the protein-bound TTQ in MADH
and the model compound may be interpreted in the context of the known crystal structure of MADH (17, 18). These results raise important questions. (i) How do the protein environments of MADH and AADH effect
the redox potential of TTQ? (ii) Why is the fully reduced quinol in the
enzyme only protonated on one hydroxyl? (iii) What is the significance
of the anionic quinol to the reaction mechanisms of these enzymes?
The fact that the redox potential of AADH is more negative than that of
MADH indicates that there must be some differences in the way the
respective proteins interact with TTQ. It was reported (16) that the
dihedral angle of the two indole rings of TTQ could influence its redox
properties. It is possible that the polypeptide could influence this
angle in the enzymes. The more negative redox potential of AADH may
also suggest that the cofactor resides in a less polar environment than
it does in MADH. A polar environment would favor the more highly
charged redox state (i.e. reduced). It is likely that the
active site of AADH will be at least somewhat more hydrophobic than
that of MADH based on their substrate specificities. MADH prefers
methylamine (30), and AADH prefers phenylethylamines (9). A less polar
environment in the active site of AADH may also explain why the anionic
semiquinone is less stable (i.e. disproportionates more
rapidly) in AADH than in MADH. The conclusion that the semiquinone in
MADH and AADH is unprotonated is consistent with results obtained with
the TTQ model compound (16). This conclusion is also reasonable because semiquinones typically exhibit pKa values <6. These
results also support the results of electron nuclear double resonance and electron spin echo envelope modulation studies of the
dithionite-generated MADH semiquinone (31). From those studies, it was
concluded that both oxygens of the radical were unprotonated and that
the radical was negatively charged.
The finding of the pH dependence of the disproportionation reaction of
MADH and the conclusion that the semiquinone/reduced couple is linked
to a proton transfer help to explain previously reported observations
of the reactivity of MADH with amicyanin in the crystalline state.
Single crystal polarized absorption spectroscopy of a binary protein
complex of MADH and amicyanin revealed that the extent to which reduced
MADH transferred an electron to amicyanin was dependent on pH (32).
Since the proteins are in complex in the crystal, after reduction of
MADH one electron is transferred to amicyanin to yield an MADH
semiquinone-reduced amicyanin complex, and the proteins can react no
further. It was observed that the amount of MADH that was present as
semiquinone rather than reduced and the amount of amicyanin that was
present as reduced rather than oxidized increased with the pH in which the crystals were incubated. These results suggested that the difference in redox potentials for the TTQ semiquinone/reduced and
amicyanin reduced/oxidized couples was pH-dependent. Our
results here are consistent with and provide an explanation for those observations.
Redox studies of the TTQ model compound indicate that the two-electron
reduction of the quinone to the quinol is linked to the transfer of two
protons. The reduced TTQH2 model compound exhibited a
pKa value of 10.1. This value is in the range of
what one would expect for a phenolic hydroxyl group. This indicates that the protein environment in MADH and AADH is somehow responsible for preventing the protonation of one of the quinol hydroxyls of TTQ in
the enzyme. An explanation for this may be inferred from inspection of
the crystal structure of MADH (Fig. 7).
The structure, which is of the oxidized form of MADH, indicates that only the C-6 carbonyl is accessible to solvent in the active site. The
C-7 carbonyl is not, and it is hydrogen-bonded to an amide hydrogen on
the peptide backbone. These results indicate that the crystal structure
presents an accurate view of the solvent accessibility of TTQ in MADH.
Since the crystal structure is of the oxidized form of MADH, these
results also suggest that the inaccessibility of the C-7 of TTQ to
solvent does not change when the enzyme is reduced. The crystal
structure of AADH has not yet been determined. These results indicate
that the solvent accessibility of only C-6 and not C-7 must be true for
AADH as well. This feature of orienting TTQ such that only one of the
quinone oxygens is accessible in the active site is apparently a common
and probably important feature that is conserved in these TTQ
enzymes.

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Fig. 7.
The active site of MADH. The TTQ
cofactor is black, and residues that surround the cofactor
are indicated. The dashed lines indicate hydrogen
bonding interactions between indole nitrogens on TTQ with main chain
oxygen atoms of Ala103 and Ser30 and between
the O-7 of TTQ and the amide nitrogen of Asp32. The O-6 is
present at the end of a solvent-accessible channel and within hydrogen
bonding distance of side chain oxygens of Asp76 and
Thr122.
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The finding that the reduced form of the enzyme-bound TTQ is anionic is
unusual and interesting. Studies of model compounds of the quinoprotein
cofactors, TTQ (16), pyrroloquinoline quinone (33), and topaquinone
(34), each reveal that the interconversion of the fully oxidized and
fully reduced forms is a two proton per two electron process. The same
is true for ubiquinone (12). The ability of TTQ enzymes to stabilize
the anionic singly protonated quinol may be relevant to the reaction
mechanisms of MADH and AADH.
It was shown for MADH (27) that during the catalytic reaction cycle, a
substrate-derived amino group displaces the accessible quinone oxygen
and is covalently attached at the C-6 position of the reduced TTQ
cofactor. The physiologically relevant reaction intermediate is an
aminoquinol. Reoxidation of the aminoquinol is a two-step process,
since the electron acceptor for MADH is a copper protein that is a
one-electron carrier. Spectroscopic (35) and kinetic (36) studies
further demonstrate that the substrate-derived amino group remains
bound to the semiquinone form of TTQ, which is generated by the first
one-electron oxidation of the aminoquinol. The presence of the amino
group significantly influences the electron transfer properties of the
aminoquinol MADH relative to quinol MADH (37, 38) and the
aminosemiquinone MADH relative to semiquinone MADH (36). The ability of
the TTQ enzymes to maintain a negative charge on the C-7 oxygen in
reduced MADH is significant because it will stabilize the amino forms of the reduced cofactor against hydrolysis. The C-6 carbon in the
anionic aminoquinol will be a much less electrophilic site than it
would be in a neutral aminoquinol. In fact, in studies of the TTQ model
compound it was possible to obtain information on amino forms of the
cofactor only by incubating it in the presence of large concentrations
of ammonia to compensate for the instability of this intermediate in
the absence of the protein. Thus, the structural properties of TTQ
enzymes that allow stabilization of the anionic fully reduced state of
the cofactor appear to be a critical feature that allows TTQ to
function as a cofactor in both catalysis and electron transfer.