Magnetic Circular Dichroism and Cobalt(II) Binding Equilibrium Studies of Escherichia coli Methionyl Aminopeptidase

Abstract

Equilibrium dialysis of methionyl aminopeptidase from Escherichia coli (EcMetAP) monitored by atomic absorption spectrometry and magnetic circular dichroism (MCD) shows that the enzyme binds up to 1.1 +/- 0.1 equiv of Co(2+) in the metal concentration range likely to be found in vivo. The dissociation constant, K(d), is estimated to be between 2.5 and 4.0 microM. Analysis of the temperature and magnetization behavior of the two major peaks in the MCD spectrum at 495 and 567 nm suggests that these transitions arise from Co(2+) with different ground states. Ligand field calculations using AOMX are used to assign the 495 nm peak to Co(2+) in the 6-coordinate binding site and the 567 nm peak to Co(2+) in the 5-coordinate site. This is further supported by the fact that the binding affinity of the Co(2+) associated with the 567 nm peak is enhanced when the pH is increased from 7.5 to 9.0, consistent with having an imidazole ligand from a histidine amino acid residue. On the basis of the MCD intensities, it is estimated that, when the 5-coordinate site is fully occupied, 0.1 equiv of cobalt is in the 6-coordinate site. Even when the cobalt concentration is very low, there is a small fraction of binuclear sites in EcMetAP formed through cooperative binding between the 5- and 6-coordinate Co(2+) ions. The magnetization behavior of the 6-coordinate Co(2+) MCD peak is consistent with an isolated pseudo-Kramer doublet ground state, suggesting that the cobalt ions in the binuclear sites are not magnetically coupled.

CaII Binding Regulates and Dominates the Reactivity of a Transition‐Metal‐Ion‐Dependent Diesterase from Mycobacterium tuberculosis

“…Relevant data are illustrated and summarized in Figure S2 in the Supporting Information and (Figure 3). [12,13,[25][26][27][28][29][30] The addition of 2.5 equivalents of Co II led to near saturation of both transitions, whichi sc onsistentw ith two tightly bound metal ions with similara ffinities fort he active site (an observation that is in agreement with the ITC data;T able 2). The transition energies werea nalyzed using AOM calculations, based on angles obtained from the crystal structure (PDB 2HY1), and were compared to the experimental data (Table3).…”

“…[25,27,28] TheM CD transitions are qualitatively similar to previously reported five and six coordinate Co II species observed in other Co II -substituted enzymes and corresponding biomimetic systems. [12,13,[25][26][27][28][29][30] An analysiso ft he variable-temperature,v ariable-field (VTVH) MCD behavior of the two transitions at l = 496 and 560 nm ( Figure S3 in the Supporting Information) indicates that the metal ions are weakly ferromagnetically coupled andt hat the couplingc onstant J is doubled upon the addition of phosphate (J = 0.7 vs.1 .4 cm À1 in the absence or presence of phosphate,r espectively;T ableS1i nt he Supporting Information). Figure 3A).…”

Use of magnetic circular dichroism to study dinuclear metallohydrolases and the corresponding biomimetics

“…By using MCD it was possible to differentiate between these two sites. Adding two equivalents of Co(II) to the apoenzyme gave Larrabee et al (2004) rise to one band at 495 nm in the MCD spectrum ( Fig. 18; Hadler et al 2008).…”

“…Octahedral high-spin Co(II) has a 4 T 1g ground state, which is subject to large positive zero-field splitting (ZFS). This creates a pseudo-Kramers doublet ground state typically 100 or more wavenumbers away from the next level, so only this doublet is populated at low temperatures (Larrabee et al 2004). Magnetization isotherms arising from these doublets will overlie each other and can often be adequately fitted by use of a simple hyperbolic tangent function (Eq.…”

“…Two MCD studies of Co(II) MetAP have been reported (Larrabee et al 2004. The MCD and absorption spectra of CoCoMetAP are shown in Fig.…”