Quinoprotein alcohol dehydrogenases use the pyrroloquinoline quinone (PQQ) cofactor to catalyze the oxidation of alcohols. The catalytic cycle is thought to involve a hydride transfer from the alcohol to the oxidized PQQ, resulting in the generation of aldehyde and reduced PQQ. Reoxidation of the cofactor by cytochrome proceeds in two sequential steps via the PQQ radical. We have used a combination of electron nuclear double resonance and density functional theory to show that the PQQ radical is not protonated at either O-4 or O-5, a result that is at variance with the general presumption of a singly protonated radical. The quantum mechanical calculations also show that reduced PQQ is unlikely to be protonated at O-5; rather, it is either singly protonated at O-4 or not protonated at either O-4 or O-5, a result that also challenges the common assumption of a reduced PQQ protonated at both O-4 and O-5. The reaction cycle of PQQ-dependent alcohol dehydrogenases is revised in light of these findings.A number of Gram-negative bacteria utilize a class of dehydrogenases known as quinoproteins, which are distinct from the flavin-and nicotinamide-dependent enzymes, to catalyze the oxidation of alcohols or aldoses (1-3). The reaction is the first step in an electron transport chain that generates a proton motive force that is used to produce ATP. Several quinoproteins contain the noncovalently bound quinoid cofactor pyrroloquinoline quinone (PQQ) 2 (Fig. 1), the role of which as a potential vitamin in mammals is currently under debate (4 -6). Among this class of enzymes, methanol dehydrogenase (MDH) (7-15), quinoprotein ethanol dehydrogenase (QEDH) (16), quinohemoprotein alcohol dehydrogenase (QH-ADH) (17, 18), and soluble glucose dehydrogenase (s-GDH) (19) have been described and crystallized. Spectroscopic, biochemical, and in particular x-ray crystallographic studies have allowed great progress to be made in the understanding of the structure and function of these proteins (20 -22).The most frequently investigated PQQ-dependent alcohol dehydrogenase is MDH. This soluble enzyme is a heterotetramer of two large and two small subunits (␣ 2 ␤ 2 ) (8 -12, 14, 15). In contrast, QEDH is composed of two large subunits (␣ 2 ) (16). The common structure of the ␣-subunit is a super-barrel composed of eight radially arranged ␤-sheets, a so-called propeller fold. The PQQ cofactor bound to a Ca 2ϩ ion is buried in the interior of the super-barrel and sandwiched between the indole ring of a tryptophan residue and an eight-member disulfide ring formed from adjacent cysteine residues. In QEDH these are Cys 105 and Cys 106 (16). Two mechanisms have been proposed for the oxidation of alcohols in quinoprotein dehydrogenases, both of which begin with the PQQ in an oxidized state. Initially, an addition/elimination mechanism was proposed, a suggestion that is now considered unlikely; rather, a hydride transfer mechanism is preferred (19, 23, 24) (see Fig. 1). Following substrate binding, the reaction is initiated by amino acid (Asp (11)...

The binding pocket of the pyrroloquinoline quinone (PQQ) cofactor in quinoprotein alcohol dehydrogenases contains a characteristic disulphide ring formed by two adjacent cysteine residues. To analyse the function of this unusual structural motif we have investigated the wild-type and a double cysteine:alanine mutant of the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa by electron paramagnetic resonance (EPR) spectroscopy. Thus, we have obtained the principal values for the full rhombic g-tensor of the PQQ semiquinone radical by high-¢eld (94 GHz) EPR necessary for a discrimination of radical species in dehydrogenases containing PQQ together with other redox-active cofactors. Our results show that the characteristic disulphide ring is no prerequisite for the formation of the functionally important semiquinone form of PQQ.

Pyrroloquinoline quinone (2,7,9-tricarboxypyrroloquinoline quinone, PQQ) is one of several quinone cofactors that is utilized in a class of dehydrogenases known as quinoproteins. In this contribution, we have used continuous-wave high-field/high-frequency electron paramagnetic resonance (EPR) at 94 GHz (W-band) to study substrate binding in ethanol dehydrogenase (QEDH) from Pseudomonas aeruginosa, taking advantage of the fact that the enzyme is isolated with a substantial proportion of the PQQ cofactor in the paramagnetic semiquinone form. In the substrate-free enzyme, the principal values of the g-tensor, obtained by spectral simulation are: gx = 2.00585(2), gy = 2.00518(2), and gz = 2.00212(2), giving giso = 2.00438(2). All three principal values of the g-tensor decrease when ethanol is bound to the protein: gx = 2.00574(2), gy = 2.00511(2), and gz = 2.00207(2), giving giso = 2.00431(2). The results represent the first direct evidence for the tight binding of an alcohol to a PQQ-dependent alcohol dehydrogenase and show that ethanol also binds to the enzyme even when the PQQ cofactor is in the semiquinone form. The decrease in g is consistent with an increase in polarity in the immediate vicinity of the PQQ cofactor and probably reflects a changed geometry of the PQQ-Ca2+ complex when ethanol binds.

Binding of methanol to the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa has been studied by pulsed electron-nuclear double resonance at 9 GHz. Shifts in the hyperfine couplings of the pyrroloquinoline quinone radical provide direct evidence for a change in the environment of the cofactor when substrate is present. By performing experiments with deuteriated methanol, we confirmed that methanol was the cause of the effect. Density functional theory calculations show that these shifts can be understood if a water molecule, which is often found in x-ray structures of the active site of quinoprotein alcohol dehydrogenases, is displaced by the substrate. The difference between the binding of water and methanol is that the water molecule forms a hydrogen bond to O5 of pyrroloquinoline quinone, which the methanol, by virtue of its methyl group, does not. The results support the proposal that aspartate rather than glutamate is the catalytically active base for a hydride transfer mechanism in quinoprotein alcohol dehydrogenases.pyrroloquinoline quinone ͉ alcohol dehydrogenase ͉ density functional theory ͉ methanol ͉ electron paramagnetic resonance P yrroloquinoline quinone (PQQ, Fig. 1) is one of several quinone derivatives that function as essential cofactors in a class of enzymes known as quinoproteins (1-3). X-ray crystallographic studies have allowed great progress to be made in the understanding of the structure and function of the PQQdependent enzymes (for reviews see refs. 4-6). There is special interest in the properties of the PQQ cofactor because its possible role as a vitamin in mammals is currently under debate (7-10).The common structural feature of methanol dehydrogenase (MDH) is an ␣ 2 ␤ 2 tetrameric structure (11-17), in contrast to quinoprotein ethanol dehydrogenase (QEDH), which is a homodimeric protein (␣ 2 ) (18, 19). In both enzymes, the ␣ subunit is a superbarrel composed of eight radially arranged ␤-sheets. The PQQ cofactor forms a complex with a Ca 2ϩ ion buried in the interior of the superbarrel and is sandwiched between the indole ring of a tryptophan and an unusual eight-membered disulfide ring structure formed from adjacent cysteines (see Fig. 2).Two alternatives have been proposed for the reaction mechanism in quinoprotein dehydrogenases (20). Initially, an addition͞elimination mechanism was favored, a suggestion that is now considered unlikely (21), because a hydride transfer mechanism is preferred (22) (see Fig. 1). The PQQ is initially oxidized, and after substrate binding, reaction is initiated by amino acid base-catalyzed proton abstraction of the hydroxyl proton of the alcohol. The characteristics of an Asp-303-Glu mutant protein (numbering scheme of MDH from Methylobacterium extorquens) were considered consistent with Asp-303 acting as the catalytically active base in MDH (16). Recently, however, this widely accepted view has been challenged with the results of a molecular dynamics study presented by Reddy and Bruice (23), who suggested that Glu-177 is the catalytic ba...