Ferredoxin-mediated electrocatalytic dehalogenation of haloalkanes by cytochrome P450(cam)

The potential role of cytochrome P450(cam) in bioremediation has been exten sively studied because of its ability to carry out the nonphysiological red uctive dehalogenation of haloalkanes. The reductive dehalogenation catalyti c cycle requires the input of two reducing equivalents, typically delivered to the enzyme from NADH via putidaredoxin reductase and putidaredoxin. In this report we present evidence demonstrating that in the presence of polyl ysine, spinach ferredoxin acts as an efficient electron shuttle between an indium-doped tin oxide (ITO) electrode and substrate-bound cytochrome P450( cam). The ferredoxin-mediated electrochemical reduction of substrate-bound cytochrome P450(cam) conduces to the reductive dehalogenation of haloalkane s. Consequently, this strategy permits the replacement of NADH and cytochro me P450 reductase, both expensive and fragile species, with an electrode to catalyze dehalogenation reactions. To accomplish this goal it was necessar y to implement a system in which the electrode exchanged electrons with spi nach ferredoxin, even in the presence of an excess of cytochrome P450(cam) which possesses a reduction potential more positive than that of spinach fe rredoxin. This molecular discrimination at the electrode surface was achiev ed by capitalizing on surface electrostatic potentials typically exhibited by electron-transfer proteins. In this particular case, the positive electr ostatic potential imparted by polylysine to the electrode surface steers sp inach ferredoxin toward the electrode with an orientation optimal for heter ogeneous electron exchange, thus acting as a good promoter for its electroc hemistry. In contrast, cytochrome P450(cam) does not exchange electrons wit h the ITO electrode but instead is readily reduced in solution by accepting an electron from spinach ferredoxin. Digital simulation of the voltammetri c experiments aimed at demonstrating the reductive dehalogenation electroca talytic activity produced important insights into the mechanism of reductiv e dehalogenations carried out by cytochrome P450(cam).