Abstract
In attempting to achieve in vitro activation of a cytochrome P450 from Bacillus megaterium , we have developed an electrochemical system which utilizes a pyrene tether. The gene encoding this protein has been used as a template to construct a series of mutant proteins, each housing a single surface cysteine. The candidate amino acids for our PCR-based site directed mutagenesis were selected by careful inspection of available higher resolution 3D structure of the enzyme. These newly introduced surface-exposed sulfhydryl groups allow for targeted anchoring of the mutant enzymes to the electrode via N-(1-pyrene)iodoacetamide active groups. Our preliminary electrochemical data revealed an early unspecified electron transfer pathway from the protein surface to the catalytic heme iron located deep inside the hydrophobic core of the enzyme.
