47113-B3
Electrochemical Activation of Cytochrome P450
During the first funding period of this project our efforts focused mainly on site-directed mutagenesis of Cytochrome P450 BM3. We previously had prepared a P450 BM3 protein with a single surface cysteine at position 387 (Cys387) to which we conjugated a pyrene-terminated linker for immobilization on graphite electrodes (Udit et. al., J. Am. Chem. Soc. 2004, 126, 10218). Position 387 provides an efficient eletron-tunneling pathway into the heme center, and proteins tethered through this site exhibit very rapid electrochemical Fe3+/2+ redox conversions, with kos values ~ 650 s-1. Indeed, this rate is sufficient to support the exclusive P450-mediated 4e- reduction of dioxygen to water, bypassing electrochemical generation of "compound I" and rendering the P450/electrode assembly inactive toward the catalytic hydroxylation of substrates.
In an effort to inhibit the 4e- dioxygen reaction, we proposed to attenuate the electron-transfer (ET) rate by moving the location of the surface cysteine (and hence pyrene tether) to vary the electrode/heme electronic coupling. To date we have engineered, expressed, and characterized four novel mutants: Cys383, Cys62, Cys97, and Cys109. These mutants feature respective cysteine-thiol/heme-iron distances of 26 , 19 , 17 , and 27 . These distances can be compared to the Cys387 spacing of 19 . We have additionally labeled the Cys383 and Cys62 mutants with pyrene, and have prepared and characterized the assembly of these conjugates on both basal-plane graphite and HOPG. Each of these pyrene-labeled engineered P450's bind strongly to graphite and exhibit reversible Fe3+/2+ redox couples in the absence of dioxygen. Integrating the cyclic voltammetry responses yields average surface coverages of ~0.5 pmol/cm2—or roughly 40% monolayer coverage based on the two-dimensional footprint of the protein.
We additionally investigated the variable-scan-rate voltammetry of these assemblies in an effort to measure their standard heterogeneous ET rate constants. These data yield a kso value for Cys383 of 0.8(1) s-1 and a kso value for Cys62 of 50(5) s-1. Notably, the ET rate for Cys383 is predicted nearly exactly by simple super-exchange coupling theory (this assumes a decay constant (b) of 1 -1 and an increased tunneling distance of 7 relative to Cys387). Interestingly, the Cys62 system exhibits a kso value 10 times smaller than that for Cys387 even though the two cysteine/heme spacings are virtually identical. The P450 BM3 crystal structure reveals a "through-space" jump in the direct tunneling pathway between Cys62 and the heme center, thus accounting for smaller-than-expected electronic coupling and attenuated ET rates. Both systems exhibit catalytic dioxygen reduction at applied potentials sufficiently negative to produce the ferrous heme centers.
In addition to preparing the pyrene conjugates of the remaining Cys97 and Cys109 mutants, we are currently working on rotated-disk voltammetry studies of Cys383- and Cys62-graphite films to determine both the products (water or peroxide) and rates of catalytic dioxygen reduction exhibited by these systems. Clearly any correlation between kso and the fate of dioxygen will have important consequences in designing electrode-bound P450s that can be converted to compound I via electrochemical methods.
In addition to the pyrene-labeling efforts, we have begun to explore alternative—and ideally more robust—strategies for conjugating biologically active molecules to electrode surfaces. Specifically, we have prepared a bifunctional conjugation linker that features a maleimide unit on one end and an ethynyl group on the other. Using this linker, we have successfully immobilized a surface-cysteine-labeled bacterial NOS (via the maleimide) to an azide-terminated alkanethiol monolayer on gold using CLICK chemistry. We are particularly excited to extend this methodology to our P450 work, as simply changing the length of the azide-alkanethiol should provide a method that is both more versatile and facile than site-directed mutagenesis for tuning the ET rate.
Finally, as an outgrowth of these efforts we used similar methodologies to prepare DNA-electrode conjugates for study via scanning electrochemical microscopy (SECM). A paper describing that work was recently published in Langmuir and has been listed under the "View/Add Citation(s)" heading of this report.
