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The ubiquitous P450 cytochromes perform challenging oxidation reactions under physiological conditions regio- and stereospecifically.  This activity can be exploited in myriad fields, including among others oxidation of inert hydrocarbons for fuels (e.g., methane → methanol) and synthesis of fine chemicals (e.g., regiospecific oxidation of long-chain alkanes for surfactants).  A key challenge to capturing P450 activity in vitro involves recapitulating the in vivo electron transfer machinery, which consists of complex proteins and cofactors. 

Our research focuses on exploiting artificial electron transfer systems for electrode-driven biocatalysis by tailoring P450 from Bacillus megaterium (BM3) to function with a mediator that will bridge the electrode-heme divide.  We believe that by placing the redox cofactors in close proximity to a derivatized metal surface that confines the protein in a conformation that permits native-like solution dynamics we can electrochemically activate the catalytic pathway.  The first few months of our grant have been used to develop methods for immobilizing P450 onto modified gold surfaces by exploiting the affinity of hexahistidine tags for Ni-NTA.  We have been able to generate P450 constructs bearing C-terminal hexahistidine tags which include the heme domain, the heme-FMN fusion protein, and the holo protein.  Notably, each construct potentially yields a different access point in the native pathway:  direct iron, FMN, and FAD reduction for the heme, heme-FMN, and holo proteins, respectively.  Absorption spectra of the Fe^II-CO species indicate that all constructs are stable (λ_max = 448 nm, no evidence of P420).  Initial attempts at surface modification have involved first modifying a gold surface with dithio-bis(N-succinimidyl propionate), followed by reaction of the surface succinimide with N-(5-amino-1-carboxypentyl) iminodiacetic acid, subsequent charging with Ni⁺², and finally incubation with the protein.  Preliminary measurements appear to yield a redox couple at -200 mV (vs. AgCl/Ag) for the heme domain.  Further experiments will involve detailed characterization of this encouraging initial result, including basic electrochemical measurements (e.g., pH dependence, k^o) and possible catalytic activity (e.g., electrolysis, RDE experiments).