ELECTRON-TUNNELING IN SUBSTRATE-REDUCED TRIMETHYLAMINE DEHYDROGENASE - KINETICS OF ELECTRON-TRANSFER AND ANALYSIS OF THE TUNNELING PATHWAY

The reoxidation of substrate-reduced trimethylamine dehydrogenase by the artificial electron acceptor ferricenium hexafluorophosphate was studied by stopped-flow spectroscopy. The rate constants for the two sequential one-electron transfers from the reduced 4Fe-4S center to ferricenium ions were measured, the first (k(a) = 49 s(-1)) being about 7 times greater than the second (k(b) = 7.3 s(-1)) at 20 degrees C and neutral pH. The temperature dependence of the second electron transfer was studied over the range 10-40 degrees C, and the rate constant ranged from 5.7 to 19.2 s(-1). Analysis of the temperature perturbation of kb by Marcus theory yielded values for the reorganizational energy of 1.95 eV and the electronic coupling matrix element of 0.26 cm(-1). An electron tunneling pathway distance of 13 +/- 0.7 Angstrom was calculated which correlates with the shortest pathway measured from the 4Fe-4S center to the protein surface using the crystallographic coordinates of trimethylamine dehydrogenase. Tyr-442 is implicated in facilitating electron transfer from the enzyme to ferricenium ions. The data suggest a location for the docking site on the surface of trimethylamine dehydrogenase for the physiological electron acceptor (ETF).