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The utility and importance of oxidation reactions are widely recognized. Examples in both industry and biology encompass conversion of inert hydrocarbons into fuels (e.g., methane → methanol), synthesis of fine chemicals (e.g., oxidation of long-chain alkanes for surfactants), hormone biosynthesis (e.g., androgens to estrogens), and drug (in)activation (e.g., activation of the chemotherapeutic cyclophosphamide). While advances have been made with regards to chemical catalysts for effecting such conversions, "green" biological catalysts remain the gold standard for performing oxidation reactions given their ability to function under physiological conditions with high degrees of regio- and stereospecificity. Heme biocatalysts are particularly attractive given their propensity towards dioxygen activation, with P450-type cytochromes among the best candidates for commercial exploitation. A key challenge to capturing heme oxygenase activity in vitro involves (and remains) recapitulating the in vivo electron transfer (ET) machinery, which consists of multiple complex proteins and (expensive, e.g., NADPH) cofactors.

Our research involves exploring and exploiting artificial ET systems for electrode-driven oxidation reactions using biological heme complexes. We have been able to expand our research to pursue exciting new directions beyond our original goals. First, the challenges involved with harnessing P450 activity in vitro lead us to consider alternatives. We turned to virus particles as versatile scaffolds for polyvalent display of heme centers that may be more electrochemically accessible, and therefore catalytically active. The platform afforded by bacteriophage Q-beta permitted us to display poly-histidine motifs; two of the constructs generated, Q-beta-Cys-His₆ and Q-beta-His₆-His₆, bind heme (absorbances at 416 and 418 nm, respectively) and were found to be electrochemically active. Experiments with rotated-disk electrodes yielded significant activity of the constructs towards dioxygen reduction, however in the presence of substrates no oxidized products were detected. Based on our prior insights from work with P450 we believe that it should be possible to fine tune the activity to alter the products of dioxygen reduction, hence influencing the catalytic activity of the virus-heme constructs. For example, mutation of the virus capsid guided by molecular modeling can lead to incorporating hydrogen bonding residues adjacent to the heme-binding moieties, which can modulate catalytic activity. We are currently at the modeling stage, with mutations and protein expression soon to follow.

Second, we continue to delve into electrode-driven P450 biocatalysis. We have been working specifically with P450 from Bacillus megaterium (BM3) which contains hydroxylase and flavin-containing reductase domains on a single polypeptide. The complex ET involved in the catalytic cycle caused us to look more closely at electrochemically accessible pathways using only the heme domain, allowing us to correlate rates of heme reduction with ET pathways and resulting product distributions. We utilized single-surface cysteine mutants of the heme domain of BM3 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 E_1/2 values (-335 to -340 mV vs AgCl/Ag). Respective ET rates (k_s^o) and heme-cysteine distances for mutants 62, 383, and 387 are 50 s^-1 and 16 Ǻ, 0.8 s^-1 and 25 Ǻ, and 650 s^-1 and 19 Ǻ, respectively. 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 k_s^o values correlating with more electrons transferred per dioxygen during catalysis. These findings will be used to guide efforts to either label the enzyme with an electron relay (to effect electrode→relay→heme ET, with enzyme in solution), or for direct bonding of the enzyme to an electrode.

We have recently established a collaboration (Dr. Jeff Warren, Caltech) to investigate the catalytic potential of yeast cytochrome c peroxidase (CCP). CCP has been extensively characterized, providing a plethora of information regarding biochemistry and activity. The enzyme is known to form stable ferryl species (high-valent iron-oxo complexes) that are catalytically active, while perturbating ferryl reactivity has been shown to be possible via mutation. Further, an electron transfer pathway has been identified and can be readily manipulated. Based on this known pathway, we hypothesize that we will be able to "wire" CCP to an electrode surface for ET to the heme, which can be oxidized in the presence of water to form reactive ferryl species that may (should) effect substrate (hydrocarbon) oxidation.

Funds provided by the ACS PRF UNI have been used almost exclusively to support student workers, all of whom have made significant contributions in the lab (e.g., sufficient data for publications and posters). This resource has allowed us to expand our research efforts, which have thus far proved to be lucrative. We hope to further develop these new projects with an emphasis on generating a range of biocatalysts displaying predictable activities that correlate with heme-protein-electrode interactions.