Designed Enzymes and the Driving Forces Behind Interdomain Electron Transfer

Abstract

Synthetic biology and biodesign approaches to redox active enzymes will require the construction of artificial electron transport chains, particularly chains which can interconvert between one- and two-electron chemistry. To both demonstrate this capability and explore the engineering parameters necessary for rapid and efficient electron transport through artificial electron transport chains, we have constructed a natural protein-designed protein chimera in which the diflavin domain of P-450 BM3 is connected to a de novo designed, heme binding four helix bundle. This single chain protein contains one FMN, one FAD, and two heme cofactors. This chimera reacts with NADPH, taking in its two electrons at the FAD cofactor, breaking them into single electrons at the FMN cofactor, and then transferring them into the artificial heme domain. We have tested three different heme analogues with varying mid-point potentials to examine the effect of driving forces on interdomain electron transfer rates. Finally, as our heme-binding domain is capable of binding oxygen in the reduced state, I will present some results using this construct as an artificial nitric oxide dioxygenase, which can perform NADPH-driven catalysis.