Zhongqing Lu

Direct electron injection from electrodes to cytochromeP450cam in biomembrane-like films

Direct, reversible, electron transfer has been demonstrated between electrodes and native cytochrome P450 cam (cyt P450 cam ) in lipid films. Electron injection directly from electrodes into the haem Fe III of cyt P450 cam has been confirmed by the influence of the known reaction of cyt P450 cam Fe II with CO on the voltammetric midpoint potential. Analysis of square wave voltammograms suggested a distribution of enzyme formal potentials in the films. With oxygen present, more than one electron was injected into the enzyme in films, mimicking in vivo electron acceptance by cyt P450Fe III and cyt P450Fe II –O 2 during catalytic oxidations. Cyt P450 cam films also catalysed electrochemically driven reduction of trichloroacetic acid in anaerobic solutions. These stable enzyme–lipid films may find applications in fundamental biochemical and toxicity studies, biocatalysis and biosensors.

Films of hemoglobin and didodecyldimethylammonium bromide with enhanced electron transfer rates

Stable thin films were made by casting vesicle dispersions of didodecyldimethylammonium bromide (DDAB) containing the protein hemoglobin (Hb) onto edge-plane pyrolytic graphite (PG) electrodes. The heme Fe(III)/Fe(II) couple of Hb had an electron transfer rate much greater in these liquid crystal films than on bare edge-plane PG electrodes or electrodes modified with mediators with Hb in solution. The formal potential was pH dependent, suggesting that one electron and one proton are involved in the reduction of Fe(III) hemes. A second quasi-reversible redox couple involving reduction of HbFe(II) was also observed in Hb-DDAB films. Trichloroacetic acid was catalytically reduced by the films at potentials of this more negative reduction. UV-vis and reflectance absorbance infrared spectra indicated that hemoglobin was not grossly denatured in the DDAB films, although conformational differences from the native state in solution are possible. Differential scanning calorimetry suggested that the films contain lamellar liquid crystalline DDAB arranged in bilayers. Electronic absorbance linear dichroism showed that Hb is preferentially oriented in the films.

Epoxidation of styrene by human cyt P450 1A2 by thin film electrolysis and peroxide activation compared to solution reactions.

Films of human cytochrome P450 1A2 (cyt P450 1A2) and polystyrene sulfonate were constructed on carbon cloth electrodes using layer-by-layer alternate absorption and evaluated for electrochemical- and H(2)O(2)-driven enzyme-catalyzed oxidation of styrene to styrene oxide. At -0.6 V vs. saturated calomel reference electrode in an electrochemical cell, epoxidation of styrene was mediated by initial catalytic reduction of dioxygen to H(2)O(2) which activates the enzyme for the catalytic oxidation. Slightly larger turnover rates for cyt P450 1A2 were found for the electrolytic and H(2)O(2) (10 mM) driven reactions compared to conventional enzymatic reactions using cyt P450s, reductases, and electron donors for cytochromes P450 1A2. Cyt P450(cam) gave comparable turnover rates in film electrolysis and solution reactions. Results demonstrate that cyt P450 1A2 catalyzes styrene epoxidation faster than cyt P450(cam), and suggests the usefulness of this thin-film electrolytic method for relative turnover rate studies of cyt P450s.

Protein-Protein Interactions in the P450 Monooxygenase System

Cytochrome bs is a small acidic hemoprotein that functions as an electron transfer protein. It is a ubiquitous mammalian membrane protein, found in the endoplasmic reticulum of most tissues. Its primary structure is highly conserved with sequence identities of greater than 89% in mammals and greater than 71% between rat and chicken (Figure I). Even in comparisons between plant (rice and tobacco) cytochrome bs with that of mammals sequence identity is greater than 35%. Twenty-five of the one hundred and thirtythree amino acids of rabbit cytochrome bs are acidic residues, glutamate or aspartate. Cytochrome bs has a number of its very highly conserved acidic residues around an exposed heme edge (Figure 2), some of which are delineated in Figure I (bold type). Residues around the exposed heme and the heme are shown in ball and stick model. It was shown quite early that cytochrome bs uses its acidic residues for stabilization of interactions with its electron transfer (redox) partners. Although not its normal redox partner, cytochrome c has been used to study electron transfer with cytochrome b5, and, since the crystal structure of both proteins have been elucidated,I-3 surface maps of the topologies of cytochrome bs and cytochrome c were constructed and fitted together making use of the invariant anionic and cationic charges respectively, ringing the exposed heme prosthetic groups as a means for determining the docking domains of the two proteins. 3 Interaction models between cytochrome b5 and methemoglobin have likewise been constructed which make use of charge pairings. 4 Interaction between erythrocyte cytochrome bs and its reductase was greatly inhibited by increased ionic strength,S which suggested that such interaction also utilizes complementary charge pairing for efficient electron transfer.