Characterization of the Second Prosthetic Group in Methanol Dehydrogenase from Hyphomicrobium X

Procedures are described for preparing 2,7,9‐tricarboxy‐1H‐pyrrolo[2,3‐ƒlquinoline‐4,5‐diol (pyrrolo‐quinoline quinol) from 2,7,9‐tricarboxy‐1H‐pyrrolo[2,3‐ƒ]quinoline‐4, 5‐dione (pyrrolo‐quinoline quinone). When methanol dehydrogenase is denatured, two compounds are liberated which have the same properties as the quinone and quinol mentioned above. On analysing the extract by high‐performance liquid chromatography, one molecule of the quinone and one molecule of the quinol per enzyme molecule are found. Mixtures of pyrrolo‐quinoline quinone and pyrrolo‐quinoline quinol at high pH produce the semiquinone form and, under certain conditions, a diamagnetic complex. Since electron spin resonance (ESR) shows that methanol dehydrogenase contains the semiquinone and the absorption spectrum suggests the presence of a diamagnetic dimer, it is tentatively concluded that the two prosthetic group molecules in the enzyme interact with each other. NMR experiments of pyrrolo‐quinoline quinone in HO demonstrate that it is partly hydrated, most prohably at the C‐5 position. Although methanol adds in the same way, it is still questionable whether the product of this addition plays a role in the mechanism of the enzymic reaction. Potentiometric titrations show a midpoint potential of the quinone/quinol couple of + 90 mV at pH 7.0 and the information of the semiquinone as an intermediate in the titration at pH 13.0. Copyright © 1981, Wiley Blackwell. All rights reserved