Structure and stability of the molybdenum cofactor intermediate cyclic pyranopterin monophosphate

Hydrogenated (reduced) pterins are found in all living organisms, where they are involved in key metabolic processes. Molybdenum in its biologically active form is bound to a fully reduced tetrahydropyranopterin referred to as a metal-binding pterin (MPT), forming the so-called molybdenum cofactor (Moco). Cyclic pyranopterin monophosphate (cPMP) is the first isolatable intermediate in molybdenum cofactor biosynthesis. Here we present for the first time a 13C NMR characterization of an active Moco intermediate. The 13C NMR data for cPMP corroborate previous data showing the tetrahydropyranopterin nature of cPMP and the presence of a gem-diol in the C1′ position of the side chain. The stability of the gem-diol, together with the absence of any observable signal at low field (175–220 ppm), is an indication that the gem-diol is not a chemical artifact, but is chemically stable and not in equilibrium with the keto form. Finally, we have studied spectrophotometrically the kinetics of cPMP oxidation in the presence of metal centers, chelating agents, and different buffers and pH values. We found that oxidation is metal-dependent and can be substantially retarded in the presence of EDTA.