In addition to the nicotinamide- and flavin-dependent dehydrogenases, there is a third class of dehydrogenases called quinoproteins that contain the novel cofactor pyrroloquinoline quinone (PQQ). While PQQ and quinoproteins were first characterized in methylotrophic bacteria, they are now recognized to be widely distributed in nature. In addition to the quinoprotein dehydrogenases, PQQ has been identified as the cofactor in the copper-containing amine oxidases which couple the oxidation of primary amines with the reduction of O2 to H2O2; PQQ-containing amine oxidases have been identified in bacteria, plants, and mammals. In addition, recent data indicate that PQQ is a nutritional requirement in mice. Clearly, PQQ and quinoproteins play a diverse and important role in nature. In methylotrophic bacteria, the PQQ-dependent methanol dehydrogenases (MDH) donate electrons from the oxidation of methanol directly through an electron transport chain to O2; this process is coupled chemi-osmotically to the net synthesis of ATP from ADP and Pi. PQQ remains enzyme bound throughout the MDH catalytic cycle and is thought to function as a 2e-/2H+ carrier and to interact directly with the primary alcohol substrate. However, there is little data on the catalytic mechanism of MDH. The overall objective of this proposal is to understand the catalytic mechanism of the PQQ-dependent MDH from Methylbacterium extorquens AM1. Specifically, experiments are proposed here that will define the role of PQQ in the oxidation of the primary alcohol substrate and identify the chemical form of PQQ-bound intermediates in the mechanism. Execution of this program will contribute to our overall understanding of the mechanism of PQQ-dependent enzymes.
