Alternate layer-by-layer polyion adsorption onto gold electrodes coated with chemisorbed mercaptopropanesulfonic acid gave stable, electroactive multilayer films containing the proteins myoglobin and cytochrome P450cam. Direct, reversible, electron exchange between gold electrodes and proteins involved heme FeIII/FeII redox couples. With oxygen in solution, electrons were also transferred to the FeII−O2 complexes of these proteins, a key step for oxidative enzyme catalysis. Film assembly for Mb was done by sequential adsorption with poly(styrenesulfonate) (PSS), DNA, or poly(dimethyl diallyl) ammonium chloride (PDDA). Cyt P450cam was assembled with layers of PSS or PDDA. Quartz crystal microbalance and voltammetric studies on the same films allowed quantitation of electroactive and nonelectroactive protein. At pH 5.5, the first protein monolayer in all films was fully electroactive. A second monolayer added 30−40% redox activity, but additional protein layers did not communicate with the electrode. Using various film construction strategies, Mb monolayers were also placed at distances from the electrodes of 0.5, 1.8, and 3.8 nm. Full electroactivity was found at 0.5 nm, and about 70−80% electroactivity at 1.8 and 3.8 nm. Results suggest the possibility of enhanced electron transport by partial intermixing of protein and nonprotein layers. Polyion films containing Mb and cyt P450cam were active for enzyme-like catalysis of styrene epoxidation in aerobic solutions.

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Protein-polyion films grown layer-by-layer and cast protein-surfactant films were employed on electrodes for catalytic oxidation of styrene derivatives to epoxides. Cytochrome P450cam and myoglobin in these films mediated the electrochemical reduction of oxygen to hydrogen peroxide, which activates these heme proteins to catalyze olefin oxidation. Compared to bare electrodes with the proteins dissolved in solution, ultrathin protein-polyion films on Au electrodes coated with mercaptopropane sulfonate gave the best catalytic activities for the oxidations. Improved performance of protein-polyion films is related to efficient, reversible heme Fe III /Fe II electron transfer and better mechanical stability than the surfactant films. Furthermore, dependence of product stereochemistry on oxygen availability in the reaction medium for the oxidation of cis--methylstyrene suggested two pathways for olefin oxidation, which had not been reported previously for cyt P450 enzymes. The stereoselective pathway depends on an active, high-valent iron-oxygen intermediate as in the natural enzyme system, while the nonstereoselective pathway may involve a peroxyl radical near the protein surface.We previously incorporated the iron heme proteins myoglobin (Mb) and cytochrome P450cam (cyt P450cam) into films of insoluble surfactants and lipids on electrodes, 1 and showed that they could be used for anaerobic electrochemical catalysis of organohalide reductions. 2,3 More recently, we constructed alternately layered films of these proteins and polyions on electrodes and demonstrated aerobic electrode-driven enzyme-like conversion of styrene to styrene oxide. 4 These reactions are "doubly catalytic", featuring mediated electrochemical reduction of protein-bound dioxygen to hydrogen peroxide, followed by peroxide-initiated, enzyme-catalyzed oxidation of the olefinic bond of styrene. Facilitation of reversible electrochemical heme Fe III /Fe II conversion is a key to the success of both types of films.Cytochrome P450 enzymes in human liver metabolize lipophilic pollutants and drugs, often to toxic products. [5][6][7][8][9] The natural catalytic cycle includes binding of substrate to cyt P450Fe III , followed by reduction to cyt P450Fe II , which binds dioxygen. The resulting cyt P450Fe II -O 2 is reduced by a second electron and ultimately forms an active iron-oxo complex which transfers oxygen to the substrate. Electrons for the natural catalytic cycle of cytosolic cytochrome P450cam in Pseudomonas putida are supplied by NADH via the enzymes putidaredoxin reductase and putidaredoxin. While camphor is the natural substrate of cytochrome P450cam, it also oxidizes camphor analogues 10-13 and other substrates such as styrenes, 14,15 alkylbenzenes, 16 tetralone, 17 and tetralin. 18 Styrene is epoxidized by cytochrome P450cam to styrene oxide, 19,20 which can damage DNA. [19][20][21] When activated by hydrogen peroxide, Mb catalyzes oxidations via a ferrylmyoglobin ( • MbFe IV dO) radical. 22 †

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Stable, functional composite films were made from the ionomer Nafion, water-insoluble surfactants, and heme proteins or the enzyme alcohol dehydrogenase. Rates of electron transfer between myoglobin (Mb) and hemoglobin (Hb) and edge plane pyrolytic graphite (PG) electrodes in these composite films were much larger than those for Hb and Mb in solution on Nafion-coated or bare PG electrodes. Films containing Nafion, didodecydimethylammonium bromide (DDAB), and hemoglobin or myoglobin retained about 90% of the initial redox activity during 4 weeks of storage at 4°C. DDAB is arranged in lamellar liquid crystal bilayers in these films at 25°C, which were more stable than films of the proteins and DDAB alone. Films of Nafion-lecithin-Cyt c on edge plane PG also had good electrochemical properties but were much less stable. Electronic and vibrational spectra indicate that the heme proteins are not denatured in the composite films, but conformational differences from the native state may exist. Linear dichroism suggests that the proteins are specifically oriented in these films. After treatment with a cross-linking agent, Nafion-lipid films containing the enzyme alcohol dehydrogenase retained twice the activity of the enzyme in solution (4°C) when stored for 2 days at room temperature.

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