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

Abstract

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 ...

Explorations of time and electrochemical potential: opportunities for fresh perspectives on signalling proteins

“…Information on the spin, and hence the ligation, state of the haem iron is easier to obtain from such features when they are monitored by MCD (magnetic circular dichroism) rather than electronic absorption spectroscopy [12]. As a consequence, a three-electrode electrochemical cell was constructed in an MCD-compatible cuvette for spectroelectrochemistry of the cardiolipin complex of cytochrome c [13,14]. The working electrode was gold foil, the counterelectrode was a platinum wire and the reference electrode was a silver wire coated with AgCl.…”

“…The volume and pathlength of the spectroelectrochemical cell, together with the absorption coefficient(s) of the spectral feature(s) of interest, will determine the amount of protein that is required. The sample requirements will be several orders of magnitude greater than for protein film electrochemistry, and time-resolved data that inform on intrinsic properties of the protein can be harder to obtain due to the limitations of stirring the sample to achieve equilibration [13]. For these reasons, methods that allow for in situ spectroscopic study of adsorbed proteins are increasingly attractive, and a number of strategies are possible, as reviewed in [19].…”

Probing a Complex of Cytochromecand Cardiolipin by Magnetic Circular Dichroism Spectroscopy: Implications for the Initial Events in Apoptosis

“…Magnetic circular dichroism (MCD) spectroscopy in conjunction with in situ potentiometric control is ideally suited to address these discrepancies. , The pattern of bands in the UV-vis (250–800 nm) MCD spectrum can be used to deduce the spin and oxidation state(s) of the heme when poised at any given electrochemical potential . Furthermore, the energy of the charge-transfer transition in the near-IR (nIR, 800–2000 nm) MCD spectrum of low-spin ferric heme is diagnostic of the chemical nature of the axial ligands .…”

Photoelastic modulator non-idealities in magneto-optical polarization measurements

“…where θ is the rotation of the polarization of light, V is the Verdet constant, a wavelength-dependent material parameter, B is the magnetic field in the direction of light and L is the distance light travels through the material in the magnetic field. 29 The difference in the imaginary part of the refractive indices for left-and right-circularly polarized light manifests itself as magnetic circular dichroism (MCD) [30][31][32]…”