Direct electron injection from electrodes to cytochrome P450cam in biomembrane-like films

Zhang, Zhe; Nassar, Alaa-Eldin F.; Lu, Zhongqing; Schenkman, John B.; Rusling, James F.

doi:10.1039/a608041c

Cited by 185 publications

(184 citation statements)

References 28 publications

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“…The peak potential for WT P450cam (45 Ϯ 2 mV) is similar to that of the control protein, and decreases to Ϫ46 Ϯ 5 mV under denaturing conditions. As seen in other studies of P450 proteins in surfactant films (37), these values are Ϸ200-300 mV more positive than redox potentials determined with nonimmobilized enzymes.…”

Section: Secis Designmentioning

confidence: 49%

“…EPR spectra of samples containing 10% glycerol were recorded at 15 K on a multipurpose Bruker E680 spectrometer equipped with an Oxford CF935 helium cryostat and EN4118X-MD-4-W1 probe head (Ϸ9.73 GHz). Redox potentials were measured by square wave voltammetry at 4°C in 50 mM potassium phosphate (pH 7.4), as previously described (37,49). Catalytic activities were determined at 25°C by monitoring the rate of NADH oxidation spectroscopically or by quantitating the formation of 5-exo-hydroxycamphor by gas chromatography (30).…”

Section: Methodsmentioning

confidence: 99%

“…The redox potential of the Fe(III)/Fe(II) couple was investigated by Osteryoung-type square wave voltammetry using enzymes that were immobilized in a didodecyldimethylammonium bromide (DDAB) film on an edge-plane graphite electrode (37). The selenoprotein has a 48 mV more negative peak potential (2 Ϯ 6 mV vs. NHE) than the control protein (50 Ϯ 4 mV) (Fig.…”

Section: Secis Designmentioning

confidence: 99%

See 2 more Smart Citations

Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine

Aldag¹,

Gromov

García-Rubio

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The unique monooxygenase activity of cytochrome P450cam has been attributed to coordination of a cysteine thiolate to the heme cofactor. To investigate this interaction, we replaced cysteine with the more electron-donating selenocysteine. Good yields of the selenoenzyme were obtained by bacterial expression of an engineered gene containing the requisite UGA codon for selenocysteine and a simplified yet functional selenocysteine insertion sequence (SECIS). The sulfur-to-selenium substitution subtly modulates the structural, electronic, and catalytic properties of the enzyme. Catalytic activity decreases only 2-fold, whereas substrate oxidation becomes partially uncoupled from electron transfer, implying a more complex role for the axial ligand than generally assumed.protein engineering ͉ selenocysteine insertion sequence element ͉ selenoenzyme ͉ stop codon suppression ͉ X-ray crystallography

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Section: Secis Designmentioning

confidence: 49%

Section: Methodsmentioning

confidence: 99%

Section: Secis Designmentioning

confidence: 99%

See 1 more Smart Citation

Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine

Aldag¹,

Gromov

García-Rubio

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…The direct electron transfer study of Kazlauskaite et al (10) showed reversible electrochemical response from glassy carbon electrodes corresponding to the first of the two electron transfers to CYP101 that are required to initiate the catalytic cycle (see step e 1 of Fig. 1), whereas Zhang et al (11) showed direct electron transfer from lipid-modified pyrolytic graphite electrodes to CYP101.…”

mentioning

confidence: 99%

A direct electrode-driven P450 cycle for biocatalysis

Reipa

Mayhew²,

Vilker³

1997

Proc. Natl. Acad. Sci. U.S.A.

105

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The large potential of redox enzymes to carry out formation of high value organic compounds motivates the search for innovative strategies to regenerate the cofactors needed by their biocatalytic cycles. Here, we describe a bioreactor where the reducing power to the cycle is supplied directly to purified cytochrome CYP101 (P450cam; EC 1.14.15.1) through its natural redox partner (putidaredoxin) using an antimony-doped tin oxide working electrode. Required oxygen was produced at a Pt counter electrode by water electrolysis. A continuous catalytic cycle was sustained for more than 5 h and 2,600 enzyme turnovers. The maximum product formation rate was 36 nmol of 5-exo-hydroxycamphor͞nmol of CYP101 per min.

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“…It has also been shown that the P450cam substrate binding site can be altered by site directed mutagenisis in order to change the enzymes specificity to other substrates [7,8]. Since the direct electrochemistry of this enzyme has been demonstrated [9] we now hope to build on this preliminary study by developing biosensors specific for halogenated hydrocarbons based on genetically engineered cytochrome P450 enzymes. …”

Section: Of 19mentioning

confidence: 99%

Bioelectrochemical dehalogenations via direct electrochemistry of poly(ethylene oxide)-modified myoglobin

Wright¹

1999

Electrochemistry Communications

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Catalytic reductive dehalogenation of hexachloroethane (HCE) was shown to be possible using a graphite electrode coated with a film of the poly(ester sulfonic acid) containing myoglobin (Mb). The effectiveness of the dehalogenation was limited by the solubility of HCE in aqueous solutions. In order to produce an electrode that could dehalogenate HCE in non-aqueous solutions, Mb was chemically modified by addition of poly(ethylene oxide) (PEO). The PEO-Mb/AQ29D modified electrode was found to be suitable for the reductive dehaolgenation the HCE in ethanolic solutions. The catalytic response was shown to be linearly dependent on the bulk concentration of HCE.

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Direct electron injection from electrodes to cytochrome P450cam in biomembrane-like films

Cited by 185 publications

References 28 publications

Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine

Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine

A direct electrode-driven P450 cycle for biocatalysis

Bioelectrochemical dehalogenations via direct electrochemistry of poly(ethylene oxide)-modified myoglobin

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