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-f]quinoline-4,5-diol (pyrrolo-quinoline quinol) from 2.7,9-tricarboxy-1 H-pyrrolo[2,3-f]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 2H2O demonstrate that it is partly hydrated, most probably 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 formation of the semiquinone as an intermediate in the titration at pH 13.0.