©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lifetime and Reactivity of the Veratryl Alcohol Radical Cation
IMPLICATIONS FOR LIGNIN PEROXIDASE CATALYSIS (*)

Luis P. Candeias (§) , Patricia J. Harvey (1)

From the (1)Gray Laboratory, P.O. Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, United Kingdom and the School of Biological & Chemical Sciences, University of Greenwich, London SE18 6PF, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The formation and decay of veratryl alcohol radical cation upon oxidation of veratryl alcohol by thallium (II) ions was studied by pulse radiolysis with spectrophotometric and conductometric detection. In aqueous solution at pH 3 the radical cation decays by a first order process, assigned to the deprotonation from the -carbon. On the basis of its lifetime (59 ± 8 ms) and of its ability to oxidize a polymeric dye (Poly R-478) we estimate that the radical cation can diffuse about 7 µm in an aqueous environment to act as a mediator of oxidations over long distances. However, 4-methoxymandelic acid is not oxidized by the veratryl alcohol radical cation in homogeneous solution, and the comparison with previous studies on lignin peroxidase catalysis suggests a second role for veratryl alcohol radical cation in the enzyme action: it may exist as an enzyme-bound species that has either a longer lifetime or a higher reduction potential than the free radical cation in bulk solution.


INTRODUCTION

Lignin peroxidase (EC 1.11.1.7), an extracellular heme peroxidase produced by white rot, has been shown to catalyze the depolymerization of lignin (1) as well as the oxidation of a range of compounds of relatively high redox potential (2) (for recent reviews see Refs. 3-5). Aromatic compounds, in particular the fungal secondary metabolite veratryl alcohol (VA)()(3,4-dimethoxybenzyl alcohol), have been shown to enhance the enzyme action, but their function has not yet been satisfactorily elucidated. It has been suggested (6) that veratryl alcohol is oxidized by the peroxidase to a radical cation and that this species plays a key role in the catalytic cycle by acting as a redox mediator between the enzyme and its substrates(3, 6) , or by converting compound III, an inactive enzyme intermediate formed by reaction of compound II with hydrogen peroxide, to the native (ferric) state(7, 8) . It has also ben proposed that VA (unoxidized) protects against formation of compound III by supplying a reducing equivalent that prevents accumulation of compound II(9, 10) .

Further progress in this field has been hampered by the lack of information on the lifetime and reactivity of the veratryl alcohol radical cation. NMR experiments (11) suggest its formation during the oxidation of VA by lignin peroxidase, but its detection by EPR has not been possible(12) . The radical cations of aromatic compounds can be conveniently studied by radiation chemistry methods, especially pulse radiolysis(13) . In this technique(14) , a short pulse of accelerated electrons, typically of a few nanoseconds, is used to generate a quantifiable amount of free radicals in solution, usually in the micromolar range, in less than a microsecond. The reactivity of these radicals is usually monitored spectrophotometrically, by conductometry or other detection techniques.

In the present study, using radiation chemistry techniques we were able to measure the veratryl alcohol radical cation absorption spectrum as well as its lifetime and reactivity toward two model compounds, a polymeric dye (Poly R-478) and 4-methoxymandelic acid.


MATERIALS AND METHODS

Commercial chemicals from Sigma or Aldrich of the highest purity available were used in this study. Solutions prepared with water purified with a Millipore Milli-Q system were degassed for about 30 min before irradiation with oxygen free nitrous oxide (NO) or with a mixture of NO and oxygen (5%, v/v) (from British Oxygen Company).

Pulse radiolysis with spectrophotometric detection was performed with a 4-MeV van de Graaff accelerator as described previously(15) . The doses per pulse were calibrated with NO-saturated solutions of KSCN solutions (10 mmol dm), assuming the product of the radiation chemical yield and extinction coefficient of (SCN) equal to 5.1 10 Gy cm (16). Conductance detection used conventional conductance cells with platinum electrodes (cell constant = 0.2 cm) to which a polarizing voltage 10 V/10 MHz AC was applied. Steady-state irradiation used a Co -source with an activity of about 35 TBq. Irradiations were performed at a dose rate of 7.9 Gy min, as determined by a Fricke dosimeter(17) .

Analysis of solutions used a HPLC equipped with a Hichrom Hypersil 50DS column and a Waters 490 multiwavelength detector, operating at 280 nm. Elution was achieved with a flow of 2 ml min of a mixture of 15 mmol dm potassium acetate at pH 3.8 and a gradient (10-50% in 5 min) of 75% acetonitrile.


RESULTS

The unstable ion Tl is a strong oxidant that reacts with many methoxylated aromatic compounds to yield the respective radical cations(13) . It can be conveniently generated by radiolysis of aqueous solutions of Tl saturated with nitrous oxide. Under these conditions, the hydroxyl radical is the main reactive species resulting from irradiation, and it reacts with Tl according to (18) .

On-line formulae not verified for accuracy

REACTION 1

In the present study solutions of Tl (1 mol dm) in 1 mmol dm HClO were used. This concentration of Tl is adjusted to give the maximum yield of Tl, whereas the HClO sets the pH close to that used in previous studies with lignin peroxidase. Under these conditions, the radiation-chemical yield of Tl was 0.46 µmol J, determined by monitoring the oxidation of ABTS (2,2`-azinobis(3-ethylbenzothiazoline 6-sulfonate)) to the stable radical ABTS (= 417 nm, = 34,700 dm mol cm(19) .

We have applied the reaction of radiolytically generated Tl with VA to produce the respective radical cation and study its spectroscopic properties and reactivity. The transient absorption spectrum recorded by pulse radiolysis on reaction of Tl with VA is shown in Fig. 1. The two absorption bands at 300 and 420 nm are characteristic of the absorption spectra of the radical cations of methoxylated aromatics(13) , suggesting that the veratryl alcohol radical cation was formed. The increase of absorption at 430 nm was exponential and had a rate constant proportional to the VA concentration (in the range 10-50 µmol dm), from which the second order rate constant of Fig. R2was determined (k = (5.4 ± 0.1) 10 dm mol s).


Figure 1: Absorption spectrum and decay kinetics of the veratryl alcohol radical cation. The radical cation was generated by pulse radiolysis of a NO-saturated solution of veratryl alcohol (70 µmol dm) and Tl (1 mmol dm) in 1 mmol dm HClO. The spectrum was built from measurements of the absorbance change at discrete wavelengths 100 µs after pulses of about 4 Gy. Inset a, time-dependence of the absorbance at 430 nm. Inset b, and of the conductance of the solution.




Figure R2: Reaction 2.



Similar experiments were performed with conductometric detection. Following the radiation pulse, a decrease of conductance was observed, attributable to the formation of OH in and consequent neutralization of one equivalent of H. However, the decreased conductance persisted longer than the completion of Fig. R2, which shows that the latter reaction does not generate or deplete further H, in support of the formation of VA.

In longer time scales, the absorption at 430 nm was observed to decay slowly, and the conductance returned to the prepulse value. We conclude therefore that VA decays with release of a proton, i.e. into an uncharged species that does not absorb at 430 nm. Through experiments with optical detection, we found that the rate of decay was independent both of the VA concentration (20-80 µmol dm) and of the initial concentration of radicals in the range 0.5 to 5 µmol dm (i.e. of the radiation dose in the range 1 to 10 Gy). In view of these observations, we conclude that the radical cation decays by a first order process, probably by deprotonation form the -carbon. The rate of this decay was determined as k = 17 ± 2 s, i.e. the lifetime of the veratryl alcohol radical cation, defined as the reciprocal of the rate constant, is 59 ± 8 ms.

Chromatographic analysis of identical solutions irradiated under steady-state conditions showed that veratryl aldehyde is the main stable product formed on oxidation of veratryl alcohol by Tl. From the dependence of the concentration of veratryl aldehyde on the radiation dose (see Fig. 3), it can be estimated that this product accounts for about 93% of the Tl generated by irradiation. The oxidation of the carbon-centerd radical formed in Fig. R3leads to veratryl aldehyde, and the high yield observed suggests that the solutions used in the experiments described contain a substance that is able to carry out this oxidation, probably Tl.


Figure 3: Formation of veratryl aldehyde and the bleaching of Poly R. Veratryl aldehyde formation on oxidation of veratryl alcohol (100 µmol dm) by Tl generated radiolytically in the absence (opensquares) or in the presence (solidsquares) of Poly R-478 (25 mg dm). The bleaching of Poly-R is shown by the solidcircles.




Figure R3: Reaction 3.



In order to evaluate the possibility that the veratryl alcohol radical cation may transfer its charge to polymers, we studied its reaction with Poly R-478 (hitherto referred to simply as Poly R). In a pulse radiolysis experiment, VA was generated by reaction with Tl in the presence of Poly R (up to 25 mg dm). Under these conditions the decay of VA, monitored by the absorption at 430 nm (Fig. 2a), was fast and exhibited a rate proportional to the concentration of the dye. Simultaneously, a bleaching of the dye could be observed at 548 nm (Fig. 2b) which had the same rate as the decay at 430 nm, a good indication that a reaction between the veratryl alcohol radical cation and the polymer was taking place. The second order rate constant of this reaction, determined from the linear dependence of the rate of decay at 430 or 548 nm on the Poly R concentration, is (5.1 ± 0.7) 10 dm (mol of monomer) s.


Figure 2: Reaction of the veratryl radical cation with Poly R. Transient absorption at 430 nm showing the decay of the veratryl alcohol radical cation (a) and at 548 nm showing the simultaneous bleaching of Poly R (b). Traces were recorded on pulse radiolysis of a NO-saturated solution of veratryl alcohol (100 µmol dm), Poly R (5 g dm), and Tl and HClO as in Fig. 1.



In addition, the same reaction was investigated by steady-state radiolysis experiments with similar solutions containing Poly R (25 mg dm). The bleaching of the dye was measured by the decrease of absorption at 548 nm, and the formation of veratryl aldehyde was monitored by HPLC. Pulse radiolysis experiments showed that under these conditions VA is formed initially in 85% yield relative to the Tl. However, in the steady-state experiments bleaching of the dye was observed, and no consumption of veratryl alcohol could be measured. Moreover, the yield of veratryl aldehyde was decreased to 7% of that measured in the absence of Poly R (Fig. 3).

It can therefore be concluded that VA reacts with Poly R to regenerate veratryl alcohol and bleach the polymer, presumably involving a radical cation of the latter as intermediate.

On-line formulae not verified for accuracy

REACTION 4

In a previous study, 4-methoxymandelic acid (MMA; hydroxy(4-methoxyphenyl)acetic acid) was used as a model recalcitrant substrate that could only be oxidized by lignin peroxidase if veratryl alcohol was present(6) . The one-electron oxidation of MMA yields a zwitterion that undergoes rapid elimination of carbon dioxide, and in the presence of oxygen the resulting radical is completely converted into anisaldehyde.()

In agreement with this mechanism, the oxidation of MMA by Tl in a solution saturated with a mixture of nitrous oxide and oxygen (20% v/v) yielded anisaldehyde in quantitative yield. A similar experiment was performed with a solution that contained, in addition, veratryl alcohol (1 mol dm). Under these conditions, most of the Tl formed (about 78%) reacts with veratryl alcohol, and accordingly the yield of anisaldehyde was greatly reduced (Fig. 4). In fact, the anisaldehyde formed under these conditions can be essentially accounted for by the fraction of Tl reacting with 4-methoxymandelic acid, suggesting that the veratryl alcohol radical cation did not oxidize MMA significantly. On the basis of our results, we estimate the rate constant of this reaction to be 5 10 dm mol s.


Figure 4: Inhibition of the oxidation of 4-methoxymandelic acid by veratryl alcohol. MMA (squares) is quantitatively oxidized to anisaldehyde (circles) in a solution saturated with NO + O (5%, v/v) (opensymbols), but in the presence of 1 mmol dm veratryl alcohol (1 mmol dm) the reaction is inhibited (solidsymbols). The solutions contained Tl and HClO (as in Fig. 1) and were irradiated and analyzed by HPLC.




DISCUSSION

Using the pulse radiolysis technique, we have been able for the first time to observe directly the formation and decay of the veratryl alcohol radical cation, to record its absorption spectrum (Fig. 1), and to measure its lifetime (59 ± 8 ms). Previous attempts to detect this species by EPR were unsuccessful(12) , and the lifetime reported here confirms that VA may be too short lived to be detected by that technique.

One of the reasons for suggesting an involvement of the veratryl alcohol radical cation in the lignin peroxidase-catalyzed degradation of lignin was the impossibility of a spatial interaction between the heme and the polymer. It was therefore proposed that veratryl alcohol ``redox cycles,'' i.e. the radical cation serves as an electron carrier between the polymer and the enzyme. However, the short lifetime attributed to VA until now seemed incompatible with this hypothesis. In conjunction with the Einstein-Smoluschowski equation of diffusion, the lifetime of 59 ms implies that VA can migrate about 7 µm in an aqueous environment. Obviously, this distance is sufficient to allow the oxidation of a polymer at a considerable distance from the peroxidase.

The ability of veratryl alcohol to redox cycle is well illustrated by the results with Poly R. In the chemical system used, the veratryl alcohol radical cation acted as an electron carrier between the oxidant (Tl) and the polymeric dye. Presumably, the veratryl alcohol radical cations generated by lignin peroxidase will behave similarly, at least toward Poly R. However, due to the difficulty of measuring degradation of an insoluble polymer, the reaction of VA with lignin has not been directly observed.

Different results were obtained with 4-methoxymandelic acid; no electron transfer between this compound and the veratryl alcohol radical cation could be observed. This is in agreement with the prediction that 4-methoxymandelic acid, with a single methoxy substituent, has a higher redox potential than veratryl alcohol. However, in the event of reversible electron transfer, equilibrium 6 would be shifted to the right by the short lifetime of the MMA radical cation.

On-line formulae not verified for accuracy

REACTION 6

Therefore, the failure to observe the oxidation of MMA by veratryl alcohol radical cation may reflect not only a thermodynamic but also a kinetic barrier to . In other words, the finite lifetime of VA may be too short for the establishment of equilibrium 6.

With respect to the oxidation of 4-methoxymandelic acid, the results of radiation chemical oxidation contrast with the previous observations in the enzyme system(6) . When equimolar mixtures of 4-methoxymandelic acid and veratryl alcohol were incubated with lignin peroxidase (in the presence of hydrogen peroxide), MMA and not VA was oxidized, although MMA alone was not a substrate for the enzyme. It appears that veratryl alcohol can oxidize 4-methoxymandelic acid in the enzyme system but not when generated in aqueous solution by a chemical oxidant. In order to solve this apparent discrepancy, we suggest that the veratryl alcohol radical cation may exist as an enzyme-bound species and that the binding may overcome either the thermodynamic or the kinetic barrier to the oxidation of methoxymandelic acid. Binding of several peroxidases to their substrates has been demonstrated, although the results obtained with veratryl alcohol and lignin peroxidase are ambiguous (20). We hypothesize that veratryl alcohol binds to the enzyme and that after electron transfer to the heme, the veratryl alcohol radical cation may react with a second substrate prior to its release.

It is conceivable that the suggested binding would prolong the lifetime of the veratryl alcohol radical cation to the extent of allowing to proceed. Cases are known in which radicals that are unstable in aqueous solution become stabilized in an enzyme environment; for example, the active ribonucleotide reductase contains a long-lived tyrosine radical(21) . Alternatively, the reduction potential of the bound veratryl alcohol radical cation may be higher than when it exists free in solution and therefore make the free energy variation associated with less positive. This hypothesis is substantiated by recent calculations showing the existence of a positively charged region around the active site of lignin peroxidase (22), which would be expected to increase the reduction potential of a radical cation immersed in it.

An alternative role for the veratryl alcohol radical cation in the catalytic cycle of lignin peroxidase is the conversion of an inactive form (compound III) back into active enzyme(8) . With the 1,2,4,5-tetramethoxybenzene radical cation this reaction was directly observed(8) , but in the case of veratryl alcohol only indirect evidence can be obtained. Clearly, the possibility of a bound veratryl alcohol radical cation is not contrary to the reactivation of compound III. The lifetime of VA implies that its steady-state concentration during the enzyme cycle will be of the order of micromolar (steady-state concentration = rate of turnover/17 s). Therefore, the reaction with compound III at a rate sufficient to prevent inactivation would require a very high bimolecular rate constant. However, a veratryl alcohol radical cation bound to the enzyme active site is readily available to react with compound III.

In summary, the lifetime of the veratryl alcohol radical cation (59 ± 8 ms) is sufficient to enable it to migrate over large distances (about 7 µm in aqueous solution) and possibly act as a redox mediator. The oxidation of Poly R-478 shows that, at least with this polymer, VA can act as a redox mediator. However, 4-methoxymandelic acid is not oxidized by VA in aqueous solution, in disagreement with the conclusions from studies with lignin peroxidase. We therefore suggest that VA can exist as an enzyme-bound species and that in this state it has either a longer lifetime or a higher reduction potential than in bulk solution.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the Cancer Research Campaign. To whom correspondence should be addressed: Gray Laboratory, P.O. Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, United Kingdom. Tel.: 44-1923-82-86-11; Fax: 44-1923-83-52-10; E-mail: candeias@graylab.ac.uk.

The abbreviations used are: VA, veratryl alcohol; MMA, 4-methoxymandelic acid; Gy, gray (SI unit of radiation dose (1 Gy = 1 J kg)); HPLC, high pressure liquid chromatography.

L. P. Candeias and P. J. Harvey, manuscript in preparation.


ACKNOWLEDGEMENTS

We are grateful to Profs. P. Jones, J. Palmer, and P. Wardman for helpful discussions, to L. K. Folkes for assistance, and to Dr. B. Vojnovic and R. Locke for the development of the pulse radiolysis equipment, in particular the conductance detection.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.