h2o2-mediated oxidation of tetrahydrobiopterin: fourier transform raman investigations provide...

JOURNAL OF RAMAN SPECTROSCOPYJ. Raman Spectrosc. 2002; 33: 610–617Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jrs.887

H2O2-mediated oxidation of tetrahydrobiopterin:Fourier transform Raman investigations providemechanistic implications for the enzymaticutilization and recycling of this essential cofactor

Jeremy Moore,† John M. Wood and Karin U. Schallreuter∗

Clinical and Experimental Dermatology, Department of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire BD7 1DP, UK

Received 1 February 2002; Accepted 11 March 2002

The oxidation of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4) by H2O2 was examined by Fouriertransform Raman spectroscopy. Initial investigations indicated that oxidation proceeds by incorporationof the H2O2 into the 6BH4 molecule without the formation of any additional water. In addition, thepyrimidine ring is affected with the shift of the double bond from the N1 — C2 to the C2 — N3 position.Such rearrangements of this double bond are observed after the production of either a carbinolamineor quinonoid species. Using deuterium exchange experiments, it was possible to substantiate that theoxidation of 6BH4 initially proceeds by the formation of a 4a-OH-carbinolamine intermediate prior toits spontaneous dehydration yielding the quinonoid dihydro species (qBH2). Furthermore, the hydrogenon the hydroxyl group of the carbinolamine interacts with the oxygen of the carbonyl group at the C4

position of the pyrimidine ring. It is proposed that this interaction facilitates the dehydration of thecarbinolamine, thus explaining its instability. Furthermore, a mechanism for the dehydration reaction issuggested, wherein the 4a-hydroxyl group forms an H-bond to the carbonyl group, thus making the oxygenof the hydroxyl group more susceptible to attack by the proton at position N5 of the pyrazine ring, resultingin qBH2 production concomitant with the loss of a water molecule. Upon increasing the concentration ofH2O2 the qBH2 converts to 7,8-BH2, which is further oxidized to L-biopterin. Taken together, our resultsdo not support an earlier proposed mechanism implicating a hydroperoxide intermediate in this oxidationreaction. Copyright 2002 John Wiley & Sons, Ltd.

INTRODUCTION

(6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (6BH4) is the es-sential cofactor for the aromatic amino acid hydroxylases[phenylalanine hydroxylase (PAH) (EC 1.14.16.1), tyrosinehydroxylase (TH) (EC 1.14.16.2) and tryptophan hydroxylase(TrpOH) (EC 1.14.16.4)] as well as the nitric oxide synthases(NOS).1,2 6BH4 also functions as an allosteric inhibitor oftyrosinase (EC 1.14.18.1), the key enzyme in the controlof pigmentation.3 Recently, 6BH4 has been found to forma stable complex with ˛-melanocyte stimulating hormone(˛-MSH). In this context, it has been suggested that thishormone–cofactor interaction could play a major role in thepigmentation process by both enzyme reactivation and by

ŁCorrespondence to: Karin U. Schallreuter, Clinical andExperimental Dermatology, Department of Biomedical Sciences,University of Bradford, Bradford, West Yorkshire BD7 1DP, UK.E-mail: [email protected]†Present address: Biomolecular Sciences, UMIST, PO Box 88,Sackville Street, Manchester M60 1QD, UK.Contract/grant sponsor: Stiefel International.

melanocortin 1 receptor signalling.4 6BH4 is recycled duringreactions catalysed by the aromatic amino acid hydroxylasesand this recycling process involves two enzymes: (a) 4a-OH-carbinolamine dehydratase (DH) (EC 4.2.1.96) catalysing thedehydration of the intermediate 4a-OH-6BH4 to quinonoiddihydrobiopterin (qBH2), and (b) dihydropteridine reduc-tase (DHPR) (EC 1.6.99.7) reducing qBH2 back to 6BH4 byutilizing NADPH as an electron donor–cofactor.5,6 In thepast, the oxidation of 6BH4 and other tetrahydropterinshas received considerable attention but the reports wereconflicting.7 – 10 However, in all cases the initial step in theoxidation pathway produced qBH2. Several pathways for thesubsequent oxidation of qBH2 have been determined usingdifferent reaction conditions yielding concomitant rearrange-ment and oxidation of the pyrazine ring and also eliminationof the dihydroxypropyl side-chain from position 6 of thepyrazine ring of 6BH4. The rearrangement of the quinonoiddihydro species yielding a dihydropterin without loss of thesubstituent in position 6 of the pyrazine ring has alreadybeen established by Archer et al.11 However, in all reports

Copyright 2002 John Wiley & Sons, Ltd.

H2O2-mediated oxidation of tetrahydrobiopterin 611

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Figure 1. FT-Raman spectra of 6BH4 (bottom) and deuterated 6BH4 (top) in the solid state.

the final product was a fully oxidized pterin ring system.Recently, the oxidation of 6BH4 has been studied with perox-ynitrite and H2O2, but the authors claimed that peroxynitriteoxidizes 6BH4 whereas H2O2 does not.12 In their experimentsEDTA was added to the reaction in order to remove tran-sition metal impurities (i.e. Cu, Mn, Fe).12 Unfortunately,this approach would yield powerful pseudocatalase activi-ties based on EDTA–transition metal complexes removingH2O2 before it could react with 6BH4.12 – 15

Here we present for the first time the Fourier transform(FT) Raman spectra of 6BH4 in aqueous solution and indeuterium oxide and also the spectra of the intermediatesand products formed during oxidation of 6BH4 by H2O2.We were able to follow the initial formation of the 4a-OH-carbinolamine from 6BH4 with increasing concentration ofH2O2. Furthermore, by utilizing deuterium isotopic shiftswe could probe the environment of the carbonyl group ofthe pyrimidine ring. These results showed that this group

Table 1. FT-Raman wavenumbers (cm�1) and assignments for 6BH4 in the solid state and in H2O and D2Osolution

Assignment6BH4

(cm�1)6BH4 in

H2O (cm�1)6BH4 in D2O

(cm�1)

C O 1687.5 1694.5 1680.0NH2 scissors bending 1659.3

1639.2 (sh)1597.21619.51597.4 1586.8

1574.8Pyrimidine ring quadrant stretch 1574.4 1560.4 1546.3Saturated pyrazine ring stretch 1527.1 1550.8 1523.8Pyrazine ring quadrant stretch 1473.8 1495.0Cyclic CH2 1454.9 1462.8 1459.9Amide vibration: 1403.7 1419.6 1384.1different orientation with 1389.6 1399.9hyperconjugation from second ringPyrimidine ring semi-circle stretch 1361.5 1363.6 1362.6

1331.0 1335.6 Shifted to 938.41309.8 1304.51226.1 1235.8

(continued overleaf )

Copyright 2002 John Wiley & Sons, Ltd. J. Raman Spectrosc. 2002; 33: 610–617

612 J. Moore, J. M. Wood and K. U. Schallreuter

Table 1. (Continued)

Assignment6BH4

(cm�1)6BH4 in

H2O (cm�1)6BH4 in D2O

(cm�1)

C–C coupled resonance with C–OH deformation 1173.8 1171.61130.31115.9 1121.61109.41093.01079.91060.2

Pyrazine ring quadrant stretch 1053.71047.11036.0 1040.71013.2 1017.8

Alkyl-substituted (6 position) pyrazine in-phase ring bend 988.1Pyrazine ring out-of-phase bend 919.8

881.4 879.9861.1810.9 810.1742.9

Pyrimidine ring out-of-plane deformation 717.6 722.0Pyrimidine ring out-of-plane deformation 690.9 686.9Pyrazine out-of-phase deformation 654.2 (sh) 659.7Pyrazine out-of-phase deformation 643.7 642.8Pyrimidine ring out-of-plane deformation: deuterated 593.2 615.6Pyrimidine ring in-plane deformation mode 551.2Pyrimidine ring in-plane deformation mode 535.2Pyrazine ring in-plane deformation mode 503.3Pyrazine ring in-plane deformation mode 486.1 489.2

also plays a role in the dehydration of the carbinolamineby H-bonding to the hydrogen of the 4a-OH group andconsequently leaving it open for attack by the proton atposition N5 of the pyrazine ring followed by elimination ofwater yielding qBH2.

EXPERIMENTAL

FT-Raman spectra were acquired using a Bruker RFS100/S spectrometer with a liquid nitrogen-cooled ger-manium detector. Near-infrared excitation was producedby an Nd3C:YAG laser operating at 1064 nm. Individualspectra were accumulated over 1000 scans at a resolu-tion of 4 cm�1 for solutions and 6000 scans at a resolu-tion of 4 cm�1 for solid samples. Spectra were recordedover the wavenumber range 500–1750 cm�1 and werecorrected for instrument response automatically withoutemploying any baseline correction techniques. For directcomparison of the intermediates formed, the spectra werestackplotted.

H2O2 (30% ACS reagent) served as a stock solu-tion and was obtained from Sigma Chemical (Gillingham,Dorset, UK). D2O2 was produced by the addition of D2O

to the stock solution of H2O2, yielding concentrationsnot less than 85% D2O2. High-purity (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin, 7,8-dihydrobiopterin and L-biopterinwere obtained from Schircks Laboratories (Jona, Switzer-land). Solid samples of the intermediates formed wereproduced by lyophilizing an aliquot of the reaction mix-ture immediately after the addition of H2O2 at pH7.0. Dried samples were stored at �80 °C prior to FT-Raman spectrum acquisition. The pH of each solution waschecked and adjusted to pH 7.0 using an Orion micro pHprobe.

RESULTS AND DISCUSSION

Recently, the FT-Raman spectra of crystalline 6BH4, BH2

and L-biopterin and their vibrational modes have beenassigned.4 Based on these results, the vibrational modesfor the intermediates and products formed by the oxidationwith H2O2 can be characterized.

We were now able to assign the vibrational shifts for6BH4 in H2O at pH 7.0 and in solution with D2O (Fig. 1 andTable 1).



Concentration-dependent oxidation of 6BH4 byH2O2

The colourless solution of 6BH4 immediately turned yellowupon addition of H2O2, indicative of the oxidation of6BH4. The FT-Raman spectra following the first five 0.1 M

additions of H2O2 clearly identified the pyrimidine ringout-of-plane deformation mode at 686.9 cm�1 increasing inintensity with increasing concentration of H2O2 (Fig. 2).In addition to the pyrimidine ring deformation modeincreasing, there is also a pronounced increase in a weakill-defined peak at 810.9 cm�1 (Fig. 2). This vibrational modeincreases dramatically with increase in H2O2 concentrationand reaches its strongest intensity upon the addition of0.4 M H2O2. Most of the low-intensity components ofthe FT-Raman spectrum are lost owing to the increased

background observed in aqueous solutions, although themajority of the ring vibrations are still present but withweaker intensities (Fig. 2). The pyrimidine and pyrazine ringquadrant stretches (1585.12 and 1558.1 cm�1) are swampedby a new feature at 1569.6 cm�1 which appears withincreasing H2O2 concentration (Fig. 2). There is an additionalnew feature produced between the other pyrazine ringquadrant stretch and cyclic CH2 vibrations (1495.6 and1463.3 cm�1) which also increases with increase in H2O2

concentration, showing a maximum at 1485.0 cm�1 (Fig. 2).The cyclic CH2 remains at a similar intensity at all times,whereas the pyrazine ring quadrant stretch is overlappedby the new features (Fig. 2). There are two new peaksthat appear in the 1300–1400 cm�1 range with increasingH2O2 concentration at 1384.2 and 1288.9 cm�1. Both peaks

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0.0

0.1

0.2

0.3

0.4

0.5

H2 O

2 (M)

686.9 810.9 1288.9 1384.2

1463.3 1485.0

1569.6 1734.7

1608.3

Figure 2. FT-Raman spectra of 6BH4 in solution (bottom) upon the addition of 0.1–0.5 M H2O2.

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0.0

0.6

0.7

0.8

H2 O

2 (M)

686.9 1307.9 1379.7 1477.0 1680.3

1602.9

1576.0

1513.9

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Wavenumber (cm-1)

1541.6

Figure 3. FT-Raman spectra of 6BH4 in solution (bottom) upon the addition of 0.6–0.8 M H2O2.



increase with similar intensity throughout the 0.1–0.5 M

range (Fig. 2). Considerable alterations are also observedin the 1500–1750 cm�1 region with two medium-intensitypeaks at 1734.7 and 1608.3 cm�1 forming a shoulder on thelow-wavenumber side of the C O stretch vibration (Fig. 2).

With H2O2 concentrations above 0.5 M, there are increasesin intensity in the pyrimidine ring out-of-plane deformationmode and also new peaks at 1307.9, 1379.7, 1576.0 and1680.3 cm�1 (Fig. 3). At 0.7 M H2O2, the new peak observednear the pyrimidine ring quadrant stretch and cyclicCH2 modes splits, yielding two vibrations (1513.9 and1477.0 cm�1), both of which increase in intensity uponthe addition of an extra 0.1 M H2O2 (Fig. 3). By contrast,the two previous modes decrease in intensity. Additionalvibrational modes are observed at 1541.6 and 1602.9 cm�1

(Fig. 3). The carbonyl stretch peak becomes more intenseand a new maximum peak derives from the shoulder at1680.3 cm�1 (Fig. 3). The other medium-intensity vibrationalmode previously observed at 1608.3 cm�1 decreases at H2O2

concentrations above 0.6 M and disappears at 0.8 M. Thepyrimidine ring out-of-plane deformation mode increasesfurther with the peak maximum shifting from 687.1 to689.6 cm�1 while the pyrazine ring vibration decreasesin 0.6 M H2O2 (Fig. 3). After the addition of 0.8 M H2O2

the resulting solution becomes cloudy and precipitates apale yellow product. The solid was collected and freeze-dried prior to acquisition of the FT-Raman spectrum. Thiscompound showed spectral properties similar to L-biopterinas one of the major oxidation products formed by the H2O2

reaction with 6BH4 (Fig. 4).

Concentration-dependent oxidationof 6BH4 by D2O2

In order to confirm this initial observation, deuteriumexchange experiments were conducted using deuterated

H2O2 (D2O2). For this purpose, H2O2 (30%) was dilutedto the required concentration with deuterium oxide toreplace the hydrogen atoms with deuterium, yieldingnot less than 85% D2O2 solution. The rationale for thisexperiment was that the addition of D2O2 to the 6BH4

solution would cause the deuterium in the D2O2 solutionto exchange with protons on the pterin ring, in additionto determining if the D2O2 interacts with other functionalgroups of 6BH4 to affect the corresponding molecularvibrations. Therefore, a decrease in the wavenumber ofvibrational modes associated with these exchanged protonswas expected.

The FT-Raman spectrum of the oxidation of 6BH4 inD2O2 showed a pattern almost identical with that forstandard H2O2 with identical peaks being formed in allareas (Fig. 5). The only difference was in the carbonyl regionwhere the C O stretch did not increase, as observed withH2O2. Indeed, the C O stretch is shifted from 1694.5 to1665.4 cm�1 with an increasing peak dependent on theconcentration of D2O2 (Fig. 5). The shift of the carbonylstretch wavenumber either could be a result of the deuteriumoxide replacing the water molecules in the solvation sphereof the carbonyl group, or it could be similar to the reactionof the aromatic amino acid hydroxylases where a hydroxylgroup is added to position 4a of the pterin ring system.The shoulder produced at around 1725 cm�1 supports theformation of a 4a-OH carbinolamine based on the C Nstretch of an amidine or guanidine group.16,17 The hydroxylgroup could be H-bonding to the carbonyl group, thus wherethe OH is replaced by an OD group a wavenumber shift isobserved. The D-bond to the carbonyl group would putconsiderable strain on the C4a —O bond, facilitating aneasier loss of the 4a-hydroxyl group with proton transferfrom the nitrogen in position 5 leading to the observed

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L-biopterin

Oxidised6BH4

Figure 4. Comparative FT-Raman spectra of the final oxidation product of 6BH4 after the addition of 0.8 M H2O2 (top) andL-biopterin (bottom) in the solid state.



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0.0

0.1

0.2

0.3

0.4

0.5

D2 O

2 (M)

1725.0 1665.4

Figure 5. FT-Raman spectra following the oxidation of 6BH4 in H2O with increasing concentration of D2O2. Each spectrum wasproduced from a freshly prepared solution with 0.1 M D2O2 increments.

dehydration similar to the reaction catalysed by the enzymeDH.18,19

In order to gain more information on this reaction, westudied the spectrum of 6BH4 in D2O (Fig. 1). The resultsclearly show that the carbonyl group is shifted to 1680.0 cm�1

by the deuterium oxide solvation, but this was not shiftedsufficiently to account for the extra movement of the peakto 1665.4 cm�1. The observation that the carbonyl group isH-bonded to the hydroxyl group was further substantiatedby adding D2O2 to the deuterated 6BH4 solution (Fig. 6).Upon addition of 0.25 M D2O2, the carbonyl stretch at1680.0 cm�1 decreased in intensity followed by the formationof a new intense carbonyl stretch at 1636.6 cm�1 (Fig. 6). This

increased shift is a consequence of the deuterium oxidesolvation of the carbonyl group together with the H-bondof the deuterated hydroxyl group to the oxygen atom. Withincreasing concentrations of D2O2, the carbonyl group stretchwavenumber decreases, whereas the 1724.2 cm�1 featureobserved in earlier experiments increased to 1725.0 cm�1

(Fig. 6). The final end product was not L-biopterin as observedafter oxidation with 0.8 M H2O2 (Fig. 4). A new compoundwas formed. This data indicated that upon deuteration, the6BH4 molecule is unable to undergo complete oxidationof the pyrazine ring, thus resulting in the formation of astable intermediate. In order to permit the acquisition of asolid spectrum, the product formed in the D2O2 experiment

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0.0

0.25

0.5

0.75

1.0

D2 O

2 (M)

1725.0

Figure 6. FT-Raman spectra of 6BH4 in D2O upon the addition of D2O2. Each spectrum was produced from a freshly preparedsolution with 0.25 M D2O2 increments.



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1725.0

Figure 7. Solid-state FT-Raman spectrum of the intermediate(s) formed after the addition of 0.4 M H2O2 to 6BH4. Note the newshoulder at 1725 cm�1 and the increase in the C O stretch due to the removal of the solvent D2O.

was collected and freeze-dried. The FT-Raman spectrumof this solid intermediate produced vibrations derivedfrom the pterin ring system, although the pyrimidine ringdeformation mode was much more pronounced (Fig. 7).The shoulder produced at around 1725 cm�1 supports theformation of a 4a-OH-carbinolamine together with qBH2,because the vibrations in this region can be assigned tothe carbon–nitrogen double bond (C N) stretch of anamidine or guanidine group.16,17 The formation of a C2 N3

double bond together with the wavenumber shift of thepyrimidine ring to a higher wavenumber in the D2O2

exchange experiments indicated the loss of the N3 protonand explains the loss of any deuterium shift associated withthis hydrogen.

The C–H stretch region for the intermediate compoundshows features almost identical with those observed for 6BH4

(Fig. 8). This observation would support the formation of a4a-OH intermediate and its dehydration to the respectiveqBH2, where in both cases all the pyrazine ring CH groupsare conserved.

CONCLUSION

To our knowledge, the data presented here provide thefirst structural evidence for the oxidation pathway of 6BH4

by H2O2 using FT-Raman spectroscopy. The initial step inthis oxidation appears to involve electrophilic attack by6BH4 on the H2O2 molecule yielding a 4a-OH-substitutedtetrahydrobiopterin (carbinolamine) intermediate togetherwith the release of a hydroxyl anion in the presence of lowconcentrations of H2O2. The formation of the other oxidationproducts such as qBH2, 7,8-dihydropterin and L-biopterinrequires higher concentrations of H2O2. A proposed reactionpathway is shown in Fig. 9.

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Figure 8. FT-Raman spectrum of the C–H stretch region of6BH4 (top) and the intermediate formed after the addition of0.4 M H2O2 (bottom) over the wavenumber range2600–3300 cm�1. The similarity of both spectra indicates thatthe CH groups of the pyrazine ring system are conserved,indicating that the oxidation must proceed via thecarbinolamine and quinonoid forms of tetrahydro- anddihydrobiopterin.



OH

OH

N

N

N

N

O

H

H CC

H

CH3

OH OH

H

H2N

N

N

N

N

O

H

H CC

H

CH3

OH OH

HH

H2N

H2N

N

N

N

N

O

H

O

H

C C

H

CH3

OH OH

HH

H2N

N

N

N

N

O

H

C C

H

CH3

OH OH

H

H2O2

6BH4

4a-OH-6BH4q-BH2

-H2O

N

N

N

N

O

H CC

H

CH3

OH OH

H

H2N

7,8-BH2 6-biopterin

H2O2N

N

N

N

O

H CC

H

CH3

OH OH

H

H2N

H

Figure 9. Proposed mechanism for the oxidation of 6BH4 by H2O2. The initial steps are the electrophilic attack of the 6BH4 on theH2O2 to form the 4a-OH-carbinolamine and liberate a hydroxyl anion. The further steps illustrate the dehydration of the4a-OH-carbinolamine to yield qBH2 and its rearrangement to 7,8-dihydrobiopterin (7,8-BH2), followed by its concomitant oxidationby H2O2 to 6-biopterin. Both the dehydration and rearrangement rates depend on the presence of H2O2, indicating that oxidizingconditions affect the stability of these intermediates.

AcknowledgementsThis research was generously funded by Stiefel International with agrant to K. U. S. Susan Shergill typed the manuscript.

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h2o2-mediated oxidation of tetrahydrobiopterin: fourier transform raman investigations provide...

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