Spectroscopic studies of quinonoid species from pyridoxal 5'-phosphate

To establish the state of protonation of quinonoid species formed nonenzymically from pyridoxal phosphate (PLP) and diethyl aminomalonate, we have studied absorption spectra of the rapidly established steady-state mixture of species. We have evaluated the formation constant and the spectrum of the mixture of Schiff base and quinonoid species. For N-methyl-PLP a singly protonated species with a peak at 464 nm is formed from the unprotonated aldehyde and the conjugate acid of diethyl aminomalonate with a formation constant Kf of 240 M-1. The very intense absorption band with characteristic vibrational structure (most evident as a shoulder at 435 nm) is accompanied by a weaker, structured band at about 380 nm and a weak, broad band at 330 nm. We suggest that the 380-nm band may represent a tautomeric form of the quinonoid compound. Protonation of the phosphate group appears to affect the spectrum only slightly. The corresponding mixture of Schiff base and quinonoid species formed from PLP has a very similar spectrum at pH 6-7. It has a formation constant Kf of 230 M-1 and a pKa of 7.8, which must be attributed to the ring nitrogen atom. The dissociated species, which may be largely carbanionic, has a strong structured absorption band at 430 nm and a weaker one, again possibly a tautomer, in the 330-nm region. The analysis establishes that in all species a proton remains on either the phenolic oxygen or the imine nitrogen. Proton NMR spectroscopy, under some conditions, reveals only two components: free PLP and what appears to be Schiff base. However, we suggest that the latter may, in fact, be a quinonoid form, either alone or in rapid equilibrium with the Schiff base. Absorption spectra of quinonoid species formed in enzymes are analyzed and compared with the spectra of the nonenzymic species.