Redox Properties of Tryptophan Tryptophylquinone Enzymes
CORRELATION WITH STRUCTURE AND REACTIVITY*

Zhenyu Zhu and Victor L. DavidsonDagger

From the Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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
<UP>CH</UP><SUB>3</SUB><UP>NH</UP><SUB>2</SUB>+<UP>H</UP><SUB>2</SUB><UP>O</UP>→<UP>CH</UP><SUB>2</SUB><UP>O</UP>+<UP>NH</UP><SUB>3</SUB>+2<UP>e</UP><SUP><UP>−</UP></SUP>+2<UP>H</UP><SUP><UP>+</UP></SUP>
<UP><SC>Reaction 1</SC></UP>
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.

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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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,
[<UP>Total species</UP>]= (Eq. 1)
k<SUB>1</SUB>[<UP>PMS<SUB>ox</SUB></UP>]+k<SUB>2</SUB>[<UP>PMS<SUB>red</SUB></UP>]+k<SUB>3</SUB>[<UP>MADH<SUB>ox</SUB></UP>]+k<SUB>4</SUB>[<UP>MADH<SUB>red</SUB></UP>]
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.
  E=E<SUB><UP>m,7,PMS</UP></SUB>+(2.3RT/nF)<UP>log</UP>(k<SUB>1</SUB>/k<SUB>2</SUB>)−(2.3RT/nF)(<UP>pH</UP>−7.0) (Eq. 2)
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).
E=E<SUB><UP>m</UP></SUB>+(2.3RT/nF)<UP>log</UP>(k<SUB>3</SUB>/k<SUB>4</SUB>) (Eq. 3)
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 (open circle ) and the oxidative titrations (bullet ).

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 (bullet ) or ascorbate (black-square) was used as a reductant. For titrations of AADH (black-down-triangle ) tyramine was used as a reductant.

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.
<UP>TTQH</UP><SUP><UP>−</UP></SUP> ⇌ <UP>TTQ</UP><SUP>·</SUP>+<UP>H</UP><SUP><UP>+</UP></SUP>+<UP>e</UP><SUP><UP>−</UP></SUP>
<UP>TTQ</UP><SUP>·</SUP> ⇌ <UP>TTQ</UP>+<UP>e</UP><SUP><UP>−</UP></SUP>
2<UP>TTQ</UP><SUP>·</SUP>+<UP>H</UP><SUP><UP>+</UP></SUP> ⇌ <UP>TTQ</UP>+<UP>TTQH</UP><SUP><UP>−</UP></SUP>
<SC><UP>Reactions</UP> 2–4</SC>
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.
<UP>TTQH</UP><SUP><UP>−</UP></SUP> ⇌ <UP>TTQH</UP><SUP>·</SUP>+<UP>e</UP><SUP><UP>−</UP></SUP>
<UP>TTQH</UP><SUP>·</SUP> ⇌ <UP>TTQ</UP>+<UP>H</UP><SUP><UP>+</UP></SUP>+<UP>e</UP><SUP><UP>−</UP></SUP>
2<UP>TTQH</UP><SUP>·</SUP> ⇌ <UP>TTQ</UP>+<UP>TTQH</UP><SUP><UP>−</UP></SUP>+<UP>H</UP><SUP><UP>−</UP></SUP>
<SC><UP>Reactions</UP> 5–7</SC>
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.

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).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

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.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM-41574.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, University of Mississippi Medical Center, 2500 N. State St., Jackson, MS 39216-4505. Tel.: 601-984-1515; Fax: 601-984-1501; E-mail: davidson{at}fiona.umsmed.edu.

1 The abbreviations used are: MADH, methylamine dehydrogenase; TTQ, tryptophan tryptophylquinone; AADH, aromatic amine dehydrogenase; PMS, phenazine methosulfate; BTP, BisTris propane (1,3-bis[tris(hydroxymethylamino)]propane); O-quinol, reduced TTQ with oxygen bonded to C-6; N-quinol, reduced TTQ with an amino group bonded to C-6.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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