Substrate binding in quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa studied by electron-nuclear double resonance

doi:10.1073/pnas.0509667103

Substrate binding in quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa studied by electron-nuclear double resonance

*Institut für Experimentalphysik, Fachbereich Physik, Freie Universität, 14195 Berlin, Germany; and
^†Fachgebiet Technische Biochemie, Institut für Biotechnologie, Technische Universität, 13353 Berlin, Germany

Edited by Nicholas J. Turro, Columbia University, New York, NY, and approved February 14, 2006 (received for review November 7, 2005)

Abstract

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.

Footnotes

^‡To whom correspondence should be addressed. E-mail: robert.bittl{at}physik.fu-berlin.de
Author contributions: C.W.M.K., H.G., and R.B. designed research; C.W.M.K. and B.M. performed research; C.W.M.K. and R.B. analyzed data; and C.W.M.K. wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

Abbreviations:

ENDOR,

electron-nuclear double resonance;

DFT,

density functional theory;

QEDH,

quinoprotein ethanol dehydrogenase;

hfc,

hyperfine coupling;

MDH,

methanol dehydrogenase;

PQQ,

pyrroloquinoline quinone