Magnetic Circular Dichroism Spectroscopy as a Probe of Geometric and Electronic Structure of Cobalt(II)-Substituted Proteins:  Ground-State Zero-Field Splitting as a Coordination Number Indicator

Abstract

Variable-temperature magnetic circular dichroism (VT MCD) is used as a probe of the ground-state electronic structure in Co(II)-substituted liver alcohol dehyrogenase, carbonic anhydrase, carboxypeptidase, substrate and inhibitor complexes of these enzymes, and four- and five-coordinate Co(II) model complexes. VT MCD was used to determine the magnitude of the ground-state zero-field splitting (ZFS) in these samples. The four-coordinate Co(II) species had ZFS's that ranged from 2.3 to 30 cm-1 and the five-coordinate species had ZFS's that ranged from 2.7 to 98 cm-1, values which fall outside of the ranges previously suggested for distinguishing 4-coordinate and 5-coordinate Co(II) (Makinen, M. W.; Kuo, L. C.; Yim, M. B.; Wells, G. B.; Fukuyama, J. M.; Kim, J. E. J. Am. Chem. Soc. 1985, 107, 5245−5255). The magnitude of the ZFS is not very sensitive to ligand heterogeneity but can be very sensitive to bond angles. Since protein active sites are in general highly angularly distorted from ideal four- or five-coordinate geometries, ZFS is rendered useless as a sole indicator of coordination number in Co(II)-substituted proteins. The MCD and absorption (or diffuse reflectance) spectra and the values of the ZFS for the proteins and model compounds are used in angle overlap method (AOM) calculations. These calculations support the conclusion that there is significant overlap in the ranges of ZFS for four- and five-coordinate Co(II) compounds.

Spectroscopic and mechanistic studies of dinuclear metallohydrolases and their biomimetic complexes

“…For example, X-ray absorption near-edge spectroscopy data from Cd(II) thiolate and imidazole complexes provided insight into the coordination number and nature of coordinating amino acids in the Cd(II)-containing carbonic anhydrase of a marine diatom, which suggested that the active site of this protein employs a tetrahedral coordination geometry and that Cd(II) is bound to a least one cysteine residue. 94 [86][87][88]95 The magnetic exchange coupling from this μ-OH bridged complex was determined to be ferromagnetic and the results from this study can be used to distinguish μ-aqua and μ-hydroxo bridging motifs in Co(II)Co(II) enzyme systems. 86,87 Neves et al were able to synthesise, and spectroscopically and kinetically characterise a dinuclear Fe(III)-Zn(II) complex with a terminal Fe(III)-bound water molecule at a position equivalent to that of the proposed nucleophile in red kidney bean PAP.…”

“…84 Zero-field splitting, g-tensors and spin states can also be obtained, in addition to magnetic coupling interaction information for dimetallic sites, by analysing variable temperature, variable field (VTVH) data. 84,87,88 MCD studies of Co(II) 2 -GpdQ, have revealed the two distinct binding sites, a 6-coordinate site with high affinity and a 5-coordinate site with low affinity. 38,78 These studies showed that GpdQ exists as a mononuclear resting state enzyme with only the α-site occupied by metal, as evidenced by the single band in the MCD spectrum at 495 nm (typical for 6-coordinate Co(II)).…”

“…88 Band intensities can also be an indicator for the coordination number of a Co(II) ion. 88 While 4-and 5-coordinate Co(II) displays bands of substantial intensity at room temperature the transitions from 6-coordinate Co(II) are up to ten times less intense. At very low temperature, however, the latter are often more intense than 5-coordinate transitions.…”

“…169,174 Zero field splitting parameters (D) can be measured by VTVH MCD and their magnitude provides valuable information about the coordination number and geometry of paramagnetic metal ions. 88 An important parameter in dinuclear Co(II) systems is the magnetic exchange coupling ( J). Larrabee et al reported a range of dinuclear Co(II)Co(II), Co(II)Co(III) and Co(III)Co(III) complexes to provide insight into the electronic structure and reactivity of cobalt substituted EcMetAp (methionine aminopeptidase from Escherichia coli).…”

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

“…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].…”