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 (ka = 49 s-1) being about 7 times greater than the second (kb = 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 A 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).