The reconstitutive ability of the isolated prosthetic group of methanol dehydrogenase with the apoenzyme of glucose dehydrogenase and the results of electron spin resonance measurements suggest that the prosthetic group has not been modified during the isolation. This result, and the properties of the directly isolated prosthetic group and derivatives, confirm the suggestion that its structure is 2,7,9-tricarboxy-1 H-pyrrolo[2,3lf]quinoline-4,5-dione.From the activity shown by derivatives of the prosthetic group and of structural analogues in the apoenzyme test it is concluded that the o-quinone structure is essential for activity. Hence the trivial name pyrrolo-quinoline quinone would be appropriate. The testing of the analogues also shows that the pyrrolo ring and the 9-carboxylic acid group are not essential for activity as they can be replaced by a pyridinol ring and a 9-hydroxy group respectively.The determination of the molar absorption coefficient of the prosthetic group (18400 M -l cm-' at 249 nm) enables its quantitative analysis. Thus it could be established that methanol dehydrogenase contains one prosthetic group per enzyme molecule. The consequences of this result in relation to already known properties of this 'quinoprotein' dehydrogenase are discussed.As was previously shown, the prosthetic group of methanol dehydrogenase is a novel, nitrogen-containing o-quinone [I -41. Recently, based on X-ray diffraction analysis of a degradation product, a chemical structure was proposed [5]. However, no evidence was presented for the presence of the o-quinone group in the proposed structure of the prosthetic group. Moreover, the question of whether the proposed structure represents that of the prosthetic group in situ remained unanswered.In this paper, the structure of the directly isolated prosthetic group is described. Furthermore, some chemical and physical properties which are valuable for its characterization and estimation, are reported.The reconstitutive ability of the prosthetic group could be investigated as we found that glucose dehydrogenase from Acinetobacter culcoaceticus contains the same prosthetic group and a suitable apoenzyme could be prepared [6]. Derivatives of the prosthetic group and structural analogues were tested with the apoenzyme system in order to see which structuralAbbreviations. ESR, electron spin resonance; NMR, nuclear magnetic resonance; compound Ia, 2,7,9-tricarboxy-lH-pyrrolo [2, compound IIa, 2,7,fl quinoline-4,5-dihydro-4,5-diol.

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Extraction of cyclopropanol-inactivated methanol dehydrogenase (MDH) gave a mixture of two interconverting compounds. The same compounds could be prepared from 2,7,9-tricarboxy-1 H-pyrrolo[2,3-,flquinoline-4,5-dione (PQQ) and cyclopropanol using a metal oxide (e.g. Ag20) as a catalyst. Structure elucidation revealed that a (25 3-propanal adduct of PQQ is formed which is present in the extract as a diastereoisomeric mixture of the ring-closed form. Cyclopropanone gave an analogous product, while cyclopropylmethanol behaved as a substrate and was oxidized by the enzyme without ring-opening.From the work described, several arguments can be derived to reject the idea that inactivation proceeds via formation of a pair of free radicals. The mechanism probably consists of a concerted proton abstraction, rearrangement of the cyclopropoxy anion to a ring-opened carbanion and attack of the latter on the electrophilic C5 of PQQ. The measured rate of inactivation (3.7 s-') is in agreement with such a mechanism. The role of the metal oxide and the enzyme in this process is the catalysis of the addition step and possibly a positioning of the reactants. As only a sole type of quinoprotein alcohol dehydrogenase becomes inhibited, the cyclopropane derivatives studied here can be regarded as mechanism-based inhibitors.The modified PQQ in cyclopropanone-inactivated M D H is fluorescent. A fluorescent intermediate was also observed in the catalytic cycle of M D H with methanol as a substrate. Its rate of formation and decay and the strongly decreased level of fluorescence in the presence of activator are in accordance with the view that the fluorescing species is the previously found oxidized-MDH . substrate (MDH,, . S) complex. Since the decomposition of this complex requires activator and model studies have failed so far to mimic the enzyme, it seems that the combination of enzyme and activator is essential for the oxidation of the alcohol substrate.Quinoprotein (i.e. PQQ-containing) methanol dehydrogenase (MDH) has been shown to become competitively inhibited by carbonyl-group reagents like cyanide and hydroxylamine [l, 21. The possibility of obtaining a stable oxidized enzyme form in the presence of these inhibitors [I] has been most useful in the detection of an enzyme-substrate complex [3]. More recently, Abeles and coworkers have shown that cyclopropanol acts as an irreversible inhibitor for M D H [4]. Complete inhibition of the monomeric M D H they used was achieved with substoichiometric amounts of cyclopropanol, this being explained by assuming that only the PQQ-semiquinone-containing enzyme molecules are reactive with this compound. However, previous studies conducted in our laboratory have clearly shown that complete inhibition of one dimeric MDH molecule requires two molecules of cyclopropanol while the enzyme should be in its fully oxidized Enzyme. Methanol dehydrogenase (EC 1.1.99.8).form [5]. Since PQQ appeared to become modified in a 1 : 1 stoichiometry and inactivation and spectral changes (above 300nm) occurred...

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