Electroenzyme-Catalyzed Oxidation of Styrene and cis-β-Methylstyrene Using Thin Films of Cytochrome P450cam and Myoglobin

Abstract

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 †

Ultrathin polymer coatings by complexation of polyelectrolytes at interfaces: suitable materials, structure and properties

“…For these purposes, mica 75,103,104,201,252,253,281,316,323,348,386,387) and float glass 19,80,131,180,182,184,185,388,389) present alternatives. Gold coated surfaces enable electrochemical studies 86,196,205,209,211,340,354,390,391) , investigations by quartz crystal microbalance (QCM) 65,86,102,119,130,202,238,266,279,305,338,339,350,367,387,391) , and studies by surface plasmon resonance (SPR) 85,137,139,…”

“…Wet chemistry cross-linking by aldehydes has been realized 253,352,358,366) , as well as reduction of graphite oxide by hydrazine and hydrogen 324) , chemical ring closure of polyimides 164,387) , azo coupling 142,148,214) , and the intercalation of acridin orange and related dyes into DNA 20,[333][334][335]343) . Also, much interest has been given to the conversion of substrates by enzymes in ESA-films 248,256,340,[346][347][348][349] . Molecular recognition reactions were studied, too 116,149,248,337,338,350,358) .…”

Protecting Peroxidase Activity of Multilayer Enzyme-Polyion Films Using Outer Catalase Layers

“…7a,11,12 In particular, HRP and cyt P450 cam activated by peroxide have provided excellent product yields with high stereoand enantio-selectivities. [13][14][15] At the same time, Mb or HRP immobilized on electrodes demonstrates good electrocatalytic activity toward H 2 O 2 , and can be used to detect H 2 O 2 . 16 Determination of H 2 O 2 is of great significance in pharmaceutical, clinical, industrial and environmental analyses, in food processing industries, and in bioanalytical chemistry.…”

Simple design of cast myoglobin/polyethyleneimine modified electrodes

“…Surfactants have been commonly used [7][8][9] to form cast films that facilitate electron transfer between Mb and electrodes. The structure of the films obtained is governed by molecular properties and interactions which guide the self-assembly process.…”