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

Abstract

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 2H2O 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.

Kinetic Study of the Antioxidant Activity of Pyrroloquinolinequinol (PQQH2, a Reduced Form of Pyrroloquinolinequinone) in Micellar Solution

“…Therefore, the increase in absorbance at 304 nm of PQQH 2 does not follow simple firstorder kinetics, showing a sigmoid curve (see Figure 5). The λ max 1 and ε 1 values of PQQ and PQQH 2 were reported by Duine et al (34), who performed the reduction of PQQ by phenylhydrazine or H 2 in the presence of PtO 2 (see Table 1). The values of λ max 1 for PQQ and PQQH 2 are similar to those obtained in the present work.…”

“…The oxidation of benzenethiol and related thiol derivatives by PQQ was performed in 0.1 M phosphate buffer solution (containing 20% CH 3 CN, pH 6.2) under anaerobic conditions, giving corresponding disulfide compounds in high yield. PQQH 2 is unstable in buffer solution (pH 7.4) under air and easily oxidized to PQQ, as reported in previous works (33)(34)(35). Consequently, in the present work, the reduction of PQQNa 2 to PQQH 2 and the measurements of the reaction rate constants were performed under strictly deaerated and nitrogensubstituted conditions by using a Hamilton 1000 series gastight syringe and sealing cap to avoid an oxidation of PQQH 2 .…”

“…The ε i values of PQQH 2 obtained are listed in Table 1, together with that reported in a previous work (34). The absorption spectrum of PQQH 2 changed to original PQQNa 2 after 25 h; PQQH 2 was comparatively stable under strict nitrogen atmosphere.…”

Quinohaemoprotein Ethanol Dehydrogenase from Comamonas testosteroni

“…The absorption spectra of the different holo-QH-EDH containing PQQ analogues, show only differences between 300 nm and 400 nm. In contrast to other PQQ-containing enzymes [32,33], instead of one, two maxima, around 315 nm and 350 nm, are observed (Fig. 8).…”

Pyrroloquinoline Quinone-Dependent Dehydrogenases fromKetogulonicigenium vulgareCatalyze the Direct Conversion ofl-Sorbosone tol-Ascorbic Acid

“…The PQQ groups of the SNDHs were identified by the method of Duine et al (10). Prosthetic groups of the enzymes were extracted with 0.5 M NaH 2 PO 4 (pH 1.0) and 67% (wt/vol) methanol.…”