Rapid photodynamics of vitamin B₆ coenzyme pyridoxal 5′-phosphate and its Schiff bases in solution

The active form of vitamin B₆, pyridoxal 5′-phosphate (PLP), is an important cofactor for numerous enzymes in amine and amino acid metabolism. Presented here is the first femtosecond transient absorption study of free PLP and two Schiff bases, PLP-valine and PLP-a-aminoisobutyric acid (AIB), in solution. Photoexcitation of free PLP leads to efficient triplet formation with an internal conversion rate that increases with increasing pH. The measured excited-state kinetics of the PLP-valine Schiff base exhibits a dramatic deuterium dependence as a result of excited-state proton transfer (ESPT) of the Cα hydrogen in the amino acid substrate. This is consistent with formation of the key reaction carbanionic intermediate (quinonoid), which is resonance stabilized by the electron-deficient, conjugated π system of the Schiff base/pyridine ring. The transient absorption signals of the PLP-Schiff base with π-methylalanine (2-aminoisobutyric acid), which does not have a Cπ proton, lack an observable deuterium effect, verifying ESPT formation of the quinonoid intermediate. In contrast to previous studies, no dependence on the excitation wavelength of the femtosecond kinetics is observed with PLP or PLP-valine, which suggests that a rapid (<250 fs) tautomerization occurs between the enolimine (absorbing at 330 nm) and ketoenamine (absorbing at 410 nm) tautomers in solution.