Direct electrochemistry of redox proteins

a b s t r a c tMagnetic circular dichroism (MCD) spectra, at ultraviolet-visible or near-infrared wavelengths (185-2000 nm), contain the same transitions observed in conventional absorbance spectroscopy, but their bisignate nature and more stringent selection rules provide greatly enhanced resolution. Thus, they have proved to be invaluable in the study of many transition metal-containing proteins. For mainly technical reasons, MCD has been limited almost exclusively to the measurement of static samples. But the ability to employ the resolving power of MCD to follow changes at transition metal sites would be a potentially significant advance. We describe here the development of a cuvette holder that allows reagent injection and sample mixing within the 50-mm-diameter ambient temperature bore of an energized superconducting solenoid. This has allowed us, for the first time, to monitor time-resolved MCD resulting from in situ chemical manipulation of a metalloprotein sample. Furthermore, we report the parallel development of an electrochemical cell using a three-electrode configuration with physically separated working and counter electrodes, allowing true potentiometric titration to be performed within the bore of the MCD solenoid.Ó 2011 Elsevier Inc. All rights reserved.Magnetic circular dichroism (MCD) 1 spectroscopy, at ultraviolet-visible and near-infrared wavelengths (185-2000 nm), has proved to be invaluable in the study of metalloproteins containing cofactors such as heme [1][2][3], non-heme iron [4], iron-sulfur clusters [5][6][7][8], cobalt [9,10], nickel [11][12][13], and copper [14,15]. The technique measures the apparent circular dichroism (CD) induced by a magnetic field [16]. Despite similarities in the instrumentation used, the observation of MCD is not dependent on the chirality of the protein; the method is equally applicable to racemic model complexes [17]. The magnetic field will always induce signals across wavelengths at which the substance absorbs. Thus, MCD spectra contain the same electronic transitions observed in conventional absorbance spectroscopy, but the bisignate nature of the spectrum provides enhanced resolution and greater detail. This spectral detail offers an unmatched fingerprinting capability that can, for example, identify the spin and oxidation states of heme groups [1] and distinguish among the variety of iron-sulfur centers found in biological molecules [18][19][20].Metalloprotein MCD can be measured at ambient or cryogenic temperatures. The latter requires adulteration with glassing agents [16] but has generally been preferred because MCD intensity from paramagnetic centers increases dramatically at low temperature. Thus, most metalloprotein MCD reported has been measured in glasses at temperatures of approximately 4.2 K. Hemoproteins represent the significant exception, giving rise to appreciable MCD at high temperatures [1]. Thus, ambient temperature MCD is used to diagnose spin state, oxidation state, and (in the case of low-spin Fe(III) hemes) axial ligation [21].As ...

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“…3B, solid circles). This midpoint potential is in good agreement with values reported previously [25,39].…”

Section: Testing Of Electrochemical Cell By Potentiometric Titrationssupporting

confidence: 93%

Electrochemical titrations and reaction time courses monitored in situ by magnetic circular dichroism spectroscopy

Bradley

Butt

Cheesman

2011

Analytical Biochemistry

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“…ref. [6][7][8][9][10][11][12][13]. Consequently, this Perspective takes the opportunity to highlight some of the challenges and opportunities we see arising from the current state of the art in the dynamic electrochemistry of redox enzymes.…”

Section: Introductionmentioning

confidence: 99%

The relationship between redox enzyme activity and electrochemical potential—cellular and mechanistic implications from protein film electrochemistry

Gates

Kemp

et al. 2011

Phys. Chem. Chem. Phys.

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In protein film electrochemistry a redox protein of interest is studied as an electroactive film adsorbed on an electrode surface. For redox enzymes this configuration allows quantification of the relationship between catalytic activity and electrochemical potential. Considered as a function of enzyme environment, i.e., pH, substrate concentration etc., the activity-potential relationship provides a fingerprint of activity unique to a given enzyme. Here we consider the nature of the activity-potential relationship in terms of both its cellular impact and its origin in the structure and catalytic mechanism of the enzyme. We propose that the activity-potential relationship of a redox enzyme is tuned to facilitate cellular function and highlight opportunities to test this hypothesis through computational, structural, biochemical and cellular studies.

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“…14 Therefore, the electrochemical mechanism for the oxidation of proteins is suitable for simulating and explaining the oxidative damage of proteins in vitro and in vivo. With the development of electrochemical apparatus and the application of modified electrodes 15 and microelectrodes, 16 electrochemical technology has obvious advantages in study of protein oxidative damage.…”

Section: Introductionmentioning

confidence: 99%

Cyclic voltammetry: A new strategy for the evaluation of oxidative damage to bovine insulin

et al. 2010

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Research on protein oxidative damage may give insight into the nature of protein functions and pathological conditions. In this work, the oxidative damage of bovine insulin on Au electrode was investigated by cyclic voltammetry (CV). The experimental results show that there are two anodic peaks for the oxidative damage of bovine insulin, which arise from the oxidation of the exposed disulfide bond SAS CYS7A,CYS7B , forming sulfenic acid RSOH (1.20 V, vs. SCE), sulfinic acid RSO 2 H and sulfonic acid RSO 3 H (1.35 V, vs. SCE). These in vitro findings not only demonstrate the applicability of CV in simulating/evaluating the oxidative damage of nonredox proteins but also find two promising candidates (two anodic peaks) for measuring insulin.

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Direct electrochemistry of redox proteins

Cited by 682 publications

References 33 publications

Electrochemical titrations and reaction time courses monitored in situ by magnetic circular dichroism spectroscopy

Electrochemical titrations and reaction time courses monitored in situ by magnetic circular dichroism spectroscopy

The relationship between redox enzyme activity and electrochemical potential—cellular and mechanistic implications from protein film electrochemistry

Cyclic voltammetry: A new strategy for the evaluation of oxidative damage to bovine insulin

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