Covalent Addition of H2O, Enzyme Substrates and Activators to Pyrrolo‐quinoline Quinone, the Coenzyme of Quinoproteins

Abstract

The fluorescence excitation and the absorption spectrum of 2,7,9-tricarboxy-1H-pyrrolo [2,3-f]quinoline- 4,s-dione (pyrrolo-quinoline quinone, PQQ), measured in the pH range 7.0- 10.0, are quite different. However. when the temperature of the solution is lowered, the shape and maxima of these spectra become more similar. ¹H-NMR in ²H_2/0 revealed a temperature-dependent equilibrium between PQQ and a hydrated form. Evidence is presented that low temperature favours the formation of the fluorescing species which is PQQ, hydrated at the C-5 position. Even further hydration is possible since absorption, fluorescence and NMR spectroscopy of PQQ in borate buffer p^H 10.0 reveal additional hydration at the C-4 position, pointing to a dihydrate. PQQ also reacts with quinoprotein enzyme substrates and activators. Spectroscopic measurements showed the existence of 5-alkoxy-5-hydroxy-PQQ and 5-amino-5-hydroxy-PQQ in the presence of alcohols and 2 M NH₄CI, p^H 9.0, respectively. In the latter case, the existence of 5-imino-PQQ could also be demonstrated. Addition compounds with amines appear to be unstable. The amines become probably oxidized because pyrrolo-quinoline quinol (PQQH₂) was found as the reaction product. On the other hand, an addition compound containing an imino bond could be isolated after addition of urea to a PQQ solution. Spectral characteristics of PQQ and its addition compounds are presented since these data are necessary for the spectral analysis of quinoproteins and the quantitative estimation of coenzyme.