Substrate-binding in quinoprotein ethanol dehydrogenase from pseudomonas aeruginosa studied by electron paramagnetic resonance at 94 GHz.
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.
Kay
Christopher W M
CW
Institut für Experimentalphysik, Fachbereich Physik, Freie Universität Berlin, 14195 Berlin, Germany.
Mennenga
Bina
B
Görisch
Helmut
H
Bittl
Robert
R
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D016880:Anisotropy; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D000431:Ethanol / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D001426:Bacterial Proteins; D000431:Ethanol; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); D002118:Calcium