PH-DEPENDENT SEMIQUINONE FORMATION BY METHYLAMINE DEHYDROGENASE FROM PARACOCCUS-DENITRIFICANS - EVIDENCE FOR INTERMOLECULAR ELECTRON-TRANSFER BETWEEN QUINONE COFACTORS

The quinonoid confactors of Paracoccus denitrifìcans methylamine dehydrogenase exhibited a pH-dependent redistribution of electrons from the 50% reduced plus 50% oxidized to the 100% semiquinone redox form. This phenomenon was only observed at pH values greater than 7.5. The semiquinone was not readily reduced by addition of methylamine, consistent with the view that this substrate donates two electrons at a time to each cofactor during catalysis. Once formed at pH 9.0, no change in redox state from 100% semiquinone was observed when the pH was shifted to 7.5, suggesting that the requirement of high pH was for formation and not stability of the semiquinone. The rate of semiquinone formation exhibited a first-order dependence on the concentration of methylamine dehydrogenase, indicating that this phenomenon was a bimolecular process involving intermolecular electron transfer between reduced and oxidized cofactors. The rate of semiquinone formation decreased with decreasing ionic strength, suggesting a role for hydrophobic interactions in facilitating electron transfer between methylamine dehydrogenase molecules. Methylamine dehydrogenase was covalently modified with norleucine methyl ester in the presence of 1-ethyl-3-[3-(di-methylamino)propyl]carbodiimide (EDC). This modification did not affect the catalytic activity of the enzyme but greatly inhibited the intermolecular redistribution of electrons at high pH. This modification also prevented subsequent cross-linking by EDC of the large subunit of methylamine dehydrogenase to amicyanin, the natural electron acceptor for this enzyme. This suggests the possibility that a common domain of methylamine dehydrogenase may participate in stabilizing protein–protein interactions which facilitate both intermolecular electron transfer between quinone cofactors and transfer to the copper center of amicyanin. © 1990, American Chemical Society. All rights reserved.