H2O2-mediated oxidation of tetrahydrobiopterin:: Fourier transform Raman investigations provide mechanistic implications for the enzymatic utilization and recycling of this essential cofactor

The oxidation of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)) by H2O2 was examined by Fourier transform Raman spectroscopy. Initial investigations indicated that oxidation proceeds by incorporation of the H2O2 into the 6BH(4) molecule without the formation of any additional water. In addition, the pyrimidine ring is affected with the shift of the double bond from the N-1 - C-2 to the C-2 - N-3 position. Such rearrangements of this double bond are observed after the production of either a carbinolamine or quinonoid species. Using deuterium exchange experiments, it was possible to substantiate that the oxidation of 6BH(4) initially proceeds by the formation of a 4a-OH-carbinolamine intermediate prior to its spontaneous dehydration yielding the quinonoid dihydro species (qBH(2)). Furthermore, the hydrogen on the hydroxyl group of the carbinolamine interacts with the oxygen of the carbonyl group at the C-4 position of the pyrimidine ring. It is proposed that this interaction facilitates the dehydration of the carbinolamine, thus explaining its instability. Furthermore, a mechanism for the dehydration reaction is suggested, wherein the 4a-hydroxyl group forms an H-bond to the carbonyl group, thus making the oxygen of the hydroxyl group more susceptible to attack by the proton at position N-5 of the pyrazine ring, resulting in qBH(2) production concomitant with the loss of a water molecule. Upon increasing the concentration of H2O2 the qBH(2) converts to 7,8-BH2, which is further oxidized to L-biopterin. Taken together, our results do not support an earlier proposed mechanism implicating a hydroperoxide intermediate in this oxidation reaction. Copyright (C) 2002 John Wiley Sons, Ltd.