Multiple Sites for Electron Tunneling between Cytochrome <i>c</i> and Mixed Self-Assembled Monolayers

Yue, Hongjun; Schrock, Kimberly; Kirby, D. J.; Knorr, Kathryn; Switzer, Stephanie; Rosmus, and Joseph; Clark, Rose A.

doi:10.1021/jp076769g

Cited by 36 publications

(46 citation statements)

References 72 publications

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“…7. In addition, and by analogy to cytochrome c, the electron transfer rate measured (in the range of 6 -31 s Ϫ1 ) is more consistent with this orientation than if CYP2C9 were laying on the SAM (Yue et al, 2008).…”

Section: Yang Et Alsupporting

confidence: 53%

See 1 more Smart Citation

Electrocatalytic Drug Metabolism by CYP2C9 Bonded to A Self-Assembled Monolayer-Modified Electrode

Yang¹,

Kabulski²,

Wollenberg³

et al. 2009

Drug Metab Dispos

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ABSTRACT:Cytochrome P450 (P450) enzymes typically require the presence of at least cytochrome P450 reductase (CPR) and NADPH to carry out the metabolism of xenobiotics. To address whether the need for redox transfer proteins and the NADPH cofactor protein could be obviated, CYP2C9 was bonded to a gold electrode through an 11-mercaptoundecanoic acid and octanethiol self-assembled monolayer (SAM) through which a current could be applied. Cyclic voltammetry demonstrated direct electrochemistry of the CYP2C9 enzyme bonded to the electrode and fast electron transfer between the heme iron and the gold electrode. To determine whether this system could metabolize warfarin analogous to microsomal or expressed enzyme systems containing CYP2C9, warfarin was incubated with the CYP2C9-SAM-gold electrode and a controlled potential was applied.The expected 7-hydroxywarfarin metabolite was observed, analogous to expressed CYP2C9 systems, wherein this is the predominant metabolite. Current-concentration data generated with increasing concentrations of warfarin were used to determine the MichaelisMenten constant (K m ) for the hydroxylation of warfarin (3 M), which is in good agreement with previous literature regarding K m values for this reaction. In summary, the CYP2C9-SAM-gold electrode system was able to carry out the metabolism of warfarin only after application of an electrical potential, but in the absence of either CPR or NADPH. Furthermore, this system may provide a unique platform for both studying P450 enzyme electrochemistry and as a bioreactor to produce metabolites without the need for expensive redox transfer proteins and cofactors.Cytochrome P450 (P450) enzymes are important for drug metabolism in humans, accounting for ϳ75% metabolism of drugs (Williams et al., 2004). Numerous factors affect the P450 metabolism rate and the resulting metabolite structure including electron transfer, protein-protein interactions, concentration and structure of the substrate, and the source and specific means whereby the P450 was prepared (Guengerich, 2007). In practice, it is difficult to isolate the individual contributions of these factors because they are often interdependent. Thus, it is of interest to devise model platforms that can be used to independently control these parameters to monitor their respective effects on metabolism.P450 catalysis requires a constant supply of NADPH as the electron source and cytochrome P450 reductase (CPR) to deliver the electrons.In attempts to obviate the requirement of these redox partners/cofactors for catalysis, P450 enzymes have been immobilized on electrodes so that the electrode supplies electrons to drive the P450 catalytic cycle (Estabrook et al., 1996;Reipa et al., 1997;Gilardi et al., 2002) with effective electrical communication between the electrode and the enzyme being critical (Yang et al., 2008). Direct adsorption of enzymes on bare electrodes, such as gold, platinum, and graphite, results in diminished biocatalytic activity (Habermüller et al., 2000;Joseph et al., 2003;Shumy...

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Section: Yang Et Alsupporting

confidence: 53%

“…Modeling of the enzyme bonded to the SAM via the N terminus suggests that the heme group of CYP2C9 is oriented in a fashion similar to bound cytochrome c (Yue et al, 2008), and, in the latter case, bonding is not required for electron transfer (Fig. 7).…”

Section: Yang Et Almentioning

confidence: 99%

Electrocatalytic Drug Metabolism by CYP2C9 Bonded to A Self-Assembled Monolayer-Modified Electrode

Yang¹,

Kabulski²,

Wollenberg³

et al. 2009

Drug Metab Dispos

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“…A distribution of formal potentials may occur and, as described below, a distribution of heterogeneous electron transfer rate constants (k et ) may be observed. [50][51][52] No changes in the voltammetric response with continuous potential cycling (30 scans) in aqueous buffer (not shown) were observed, demonstrating the stability of the adsorbed protein.…”

Section: Voltammetric Characterization Of Cyt C Immobilized On Planarmentioning

confidence: 97%

Characterization of Nanoporous Gold Electrodes for Bioelectrochemical Applications

Scanlon¹,

Salaj-Kośla²,

Belochapkine³

et al. 2011

Langmuir

101

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ABSTRACT. The high surface areas of nanostructured electrodes can provide for significantly enhanced surface loadings of electroactive materials. The fabrication and characterisation of nanoporous gold (np-Au) substrates as electrodes for bioelectrochemical applications is described.Robust np-Au electrodes were prepared by sputtering a gold-silver alloy onto a glass support and subsequent de-alloying of the silver component. Alloy layers were prepared with either a uniform or non-uniform distribution of silver and, post de-alloying, showed clear differences in morphology on characterisation with scanning electron microscopy. Redox reactions under kinetic control, in particular measurement of the charge required to strip a gold oxide layer, provided the most accurate measurements of the total electrochemically addressable electrode surface area, A real .Values of A real up to 28 times that of the geometric electrode surface area, A geo , were obtained. For diffusion controlled reactions overlapping diffusion zones between adjacent nanopores established limiting semi-infinite linear diffusion fields where the maximum current density was dependent on A geo . The importance of measuring the surface area available for the immobilisation was determined 2 using the redox protein, cyt c. The area accessible to modification by a biological macromolecule, A macro , such as cyt c was reduced by up to 40 % compared to A real , demonstrating that the confines of some nanopores were inaccessible to large macromolecules due to steric hindrances. Preliminary studies on the preparation of np-Au electrodes modified with osmium redox polymer hydrogels and Myrothecium verrucaria bilirubin oxidase (MvBOD) as a biocathode were performed; current densities of 500 A cm -2 were obtained in unstirred solutions.

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“…with that via a covalently bound one. A widely used method to produce a monolayer of CytC is based on using its positive surface charges at neutral pH (isoelectric point ∼10.0) (26) to electrostatically adsorb it onto a carboxylate-terminated linker that, in turn, is bound covalently to the electrode (9,(14)(15)(16)(17)(18)(19)(20). For measuring the ETp via adsorbed CytC, we functionalized the surface of high-doped Si with a carboxylate (see Experimental Procedures for further details).…”

Section: Significancementioning

confidence: 99%

“…Previous studies of intramolecular electron transfer (ET) in CytC used both spectroscopic (1-6) and electrochemical measurements (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). In the latter studies, where the rate of ET between the intrinsic redox site and a working electrode has been probed, the CytC was usually attached (covalently or electrostatically) to one electrode via a linker molecule to form a monolayer.…”

mentioning

confidence: 99%

Solid-state electron transport via cytochrome c depends on electronic coupling to electrodes and across the protein

Amdursky

Ferber

Bortolotti

et al. 2014

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

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Electronic coupling to electrodes, Γ, as well as that across the examined molecules, H, is critical for solid-state electron transport (ETp) across proteins. Assessing the importance of each of these couplings helps to understand the mechanism of electron flow across molecules. We provide here experimental evidence for the importance of both couplings for solid-state ETp across the electron-mediating protein cytochrome c (CytC), measured in a monolayer configuration. Currents via CytC are temperature-independent between 30 and ∼130 K, consistent with tunneling by superexchange, and thermally activated at higher temperatures, ascribed to steady-state hopping. Covalent protein-electrode binding significantly increases Γ, as currents across CytC mutants, bound covalently to the electrode via a cysteine thiolate, are higher than those through electrostatically adsorbed CytC. Covalent binding also reduces the thermal activation energy, E a , of the ETp by more than a factor of two. The importance of H was examined by using a series of seven CytC mutants with cysteine residues at different surface positions, yielding distinct electrode-protein(-heme) orientations and separation distances. We find that, in general, mutants with electrode-proximal heme have lower E a values (from high-temperature data) and higher conductance at low temperatures (in the temperatureindependent regime) than those with a distal heme. We conclude that ETp across these mutants depends on the distance between the heme group and the top or bottom electrode, rather than on the total separation distance between electrodes (protein width).bioelectronics | temperature dependence | protein conduction

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Multiple Sites for Electron Tunneling between Cytochrome c and Mixed Self-Assembled Monolayers

Cited by 36 publications

References 72 publications

Electrocatalytic Drug Metabolism by CYP2C9 Bonded to A Self-Assembled Monolayer-Modified Electrode

Electrocatalytic Drug Metabolism by CYP2C9 Bonded to A Self-Assembled Monolayer-Modified Electrode

Characterization of Nanoporous Gold Electrodes for Bioelectrochemical Applications

Solid-state electron transport via cytochrome c depends on electronic coupling to electrodes and across the protein

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