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

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 ...

Ligation and Reactivity of Methionine-Oxidized Cytochrome c

“…54 Lysine ligation to the heme iron at near neutral pH has also been observed upon carboxymethylation 55 and tyrosine nitration of cyt c 56 and also upon binding to CL. 57 Since changes in the intraprotein hydrogen bonding and heme packing appear to facilitate the transition, we have hypothesized that structural perturbation associated with oxidation of Met80 could also favor Lys ligation to the ferric heme iron.…”

Disruption of cytochrome c heme coordination is responsible for mitochondrial injury during ischemia

“…Globin-like pentacoordinated cyt c can react with oxygen or H 2 O 2 to promote oxygenase/peroxidase activity of that enzyme [22, 23, 26]. Calcium, which is elevated during ischemia, was recently shown to potentiate peroxidase activity of cyt c [26].…”

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 cardiolipin complex of oxidized cytochrome c exists as two conformers in which the haem iron is low-spin; approximately 40% has histidine/hydroxide axial Fe(III) ligation and the remainder has histidine/lysine axial Fe(III) ligation [14]. On equilibrating the cardiolipin complex of cytochrome c at increasingly negative, and then increasingly positive, potentials, the MCD changes are fully reversible.…”