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Abstract: Developing electrode-driven biocatalytic systems utilizing the P450 cytochromes for selective oxidations depends not only on achieving electron transfer (ET) but also doing so at rates that favor native-like turnover. Herein we report studies that correlate rates of heme reduction with ET pathways and resulting product distributions. We utilized single-surface cysteine mutants of the heme domain of P450 from Bacillus megaterium and modified the thiols with N-(1-pyrene)-iodoacetamide, affording proteins that could bond to basal-plane graphite. Of the proteins examined, Cys mutants at position 62, 383, and 387 were able to form electroactive monolayers with similar E1/2 values (−335 to −340mV vs AgCl/Ag). Respective ET rates (ks o) and heme-cysteine distances for 62, 383, and 387 are 50 s-1 and 16Ǻ, 0.8 s–1 and 25Ǻ, and 650 s–1 and 19Ǻ. Experiments utilizing rotated-disk electrodes were conducted to determine the products of P450-catalyzed dioxygen reduction. We found good agreement between ET rates and product distributions for the various mutants, with larger ks o values correlating with more electrons transferred per dioxygen during catalysis. [Copyright &y& Elsevier]

Cyclic voltabsorptometry is used for the first time to distinguish and characterize electrochemically the active (P450) and inactive (P420) forms of cytochromes P450 immobilized on an electrode during voltammetry experiments. This was achieved by using the heme domain (BMP) of the bacterial cytochrome P450 BM3 from Bacillus megaterium(CYP102A1) immobilized on mesopouros tin-oxide (SnO 2) electrodes. We demonstrate that the formation of either the P450 form or the P420 one can be obtained by modifying the mesoporous electrode surface with polycations with different properties such as polyethylenimmine (PEI) and polydiallyldimethylammonium chloride (PDDA). Potential step spectroelectrochemistry allowed measurement of reduction potentials of the active P450 form. Values of −0.39 ± 0.01 V and −0.58 ± 0.01 V (both versus Ag/AgCl) were calculated for the active P450 form immobilized on the BMP/PDDA−SnO 2and BMP/PEI−SnO 2electrodes, respectively. The cyclic voltabsorptometric experiments showed how, when both the active and inactive forms are present on the PEI film, the inactive P420 species tends to dominate the cyclic voltammetric signal. [ABSTRACT FROM AUTHOR]