Open AccessThe Role of Pyrrolo‐quinoline Semiquinone Forms in the Mechanism of Action of Methanol Dehydrogenase
Author(s) -
BEER Ron,
DUINE Johannis A.,
FRANK Jzn,
WESTERLING Jaap
Publication year - 1983
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1983.tb07123.x
Subject(s) - methanol dehydrogenase , chemistry , semiquinone , electron paramagnetic resonance , quinoline , methanol , photochemistry , enzyme , electron paramagnetic resonance spectroscopy , dehydrogenase , substrate (aquarium) , crystallography , stereochemistry , redox , inorganic chemistry , organic chemistry , nuclear magnetic resonance , biology , physics , ecology
On storage of methanol dehydrogenase of Hyphomicrobium X as a solution, the electron spin resonance (ESR) spectrum becomes more complicated. Ka‐band (35 GHz) spectroscopy reveals that a paramagnetic species with a lower g ‐vale is formed from pyrrolo‐quinoline semiquinone (PQQH) during the ageing process. From the way in which this second paramagnetic species is formed and the absence of an effect of substrate, activator or both on fresh or aged enzyme, it follows that the complex ESR spectrum cannot be used as an argument to support the view that a three‐electron reduced form of PQQ exists in methanol dehydrogenase. X‐band ESR spectroscopy of an enzyme solution reveals additional peaks at g = 2.03 and g = 2.01 only when the preparation contains O 2 and is measured at liquid nitrogen temperature. The results point to the formation of a complex from PQQH and O 2 , most probably a peroxyl radical which only exists at low temperature. Direct reduction of methanol dehydrogenase, as it is isolated, is possible. The enzyme form thus produced has the same properties as that obtained from reduction of oxidized methanol dehydrogenase with substrate, containing only a low amount of PQQH‐but not other free radical species. This substantiates the earlier conclusion that the isolated enzyme is in a half‐oxidized form. A view on the mechanism of action of the enzyme is proposed which is quite opposite to the one that was recently advocated [Mincey et al. (1981) Biochemistry, 20 , 7502–7509] since former and present results lead to the following conclusions: (a) there is no evidence for the involvement of a three‐electron reduced form of PQQ in the catalytic cycle of the enzyme; (b) enzyme molecules containing free radical species are not reduced by substrate; (c) the product is released before the enzyme is oxidized by electron acceptor.