MCD spectroscopy

In a few instances the technique of magnetic circular dichroism (MCD) spectroscopy has been used to corroborate assignments based on UV-visible spectroscopy. For example, the assignment of the intense 360 nm band for [S,N,Y to a r (2e") r (2a2") (HOMO LUMO) excitation has been confirmed by the measurement of the MCD spectrum of The MCD spectrum of [S4N3] indicates that each of the... 

A. Electronic Spectroscopy Absorption, Circular Dichroism, and Magnetic Circular Dichroism (MCD) Spectroscopy... 

Using a combination of techniques such as EPR, resonance Raman, and MCD spectroscopy, the conversion of [2Fe-2S] into [4Fe—4S] centers has been found to take place under reducing conditions in E. coli biotin synthase 281). The as-prepared form of this enzyme has been thought to contain one [2Fe-2S] center per monomer, coordinated by the three cysteine residues of the motif Cys-X3-Cys-X2-Cys and by a fourth, noncysteinyl ligand. Upon reduction, a [4Fe-4S] cluster bridging two monomers may be formed in the active enzyme. In the reduced state, the [4Fe-4S] center is characterized by a mixture of S = I and S = k spin states giving EPR features at g 5.6 and... 

The spin-Hamiltonian concept, as proposed by Van Vleck [79], was introduced to EPR spectroscopy by Pryce [50, 74] and others [75, 80, 81]. H. H. Wickmann was the first to simulate paramagnetic Mossbauer spectra [82, 83], and E. Miinck and P. Debmnner published the first computer routine for magnetically split Mossbauer spectra [84] which then became the basis of other simulation packages [85]. Concise introductions to the related modem EPR techniques can be found in the book by Schweiger and Jeschke [86]. Magnetic susceptibility is covered in textbooks on molecular magnetism [87-89]. An introduction to MCD spectroscopy is provided by [90-92]. Various aspects of the analysis of applied-field Mossbauer spectra of paramagnetic systems have been covered by a number of articles and reviews in the past [93-100]. 

The Zn11 ion of angiotensin-converting enzyme (ACE) has been replaced by Co11 to give an active, chromophoric enzyme and inhibitor binding has been identified spectroscopically. Visible and MCD spectroscopy were used to characterize the catalytic metal binding site in the... 

EPR, ENDOR, Mossbauer, EXAFS, and MCD spectroscopies to further elucidate intimate details of electron and proton transfer within nitrogenase are difficult... 

MCD spectroscopy in range 300 to 2000 nm at both ambient and liquid helium (4.2 K) temperatures can yield information about the spin, oxidation, and coordination states of each heme in a multiheme protein such as CCP (75). This technique, in combination with low-temperature X-band EPR spectroscopy, was used to great effect in characterizing the properties of the fully oxidized and MV forms of the P. aeruginosa CCP in solution. At 4.2 K, both hemes in the oxidized enzyme are low-spin ferric, with diagnostic features in the near infrared-MCD (NIR-MCD) spectrum consistent with one heme with His/Met axial coordination and the other with bis-histidine axial coordination this is entirely consistent with the crystal structure. In contrast, at room temperature only the low-potential (bis-histidine coordinated) heme in the C-terminal domain remains completely low-spin, whereas the high-potential (His/Met coordinated) heme exists as mixture of high- and low-spin forms 58). 

Magnetic susceptibility studies can be difficult to interpret for multicentered metalloproteins particularly when paramagnetic impurities are present, whereas with MCD spectroscopy the magnetic properties of individual centers can be investigated independently. However, it should be emphasized that MCD studies... 

This pedagogical account is intended to provide a brief introduction for the non-specialist, to the theoretical and experimental aspects of variable temperature MCD spectroscopy that are applicable in the study of metallopro-teins. This is followed by some individual examples of MCD studies of metallo-proteins that have been chosen to illustrate the utility of the technique and the type of information that is available. 

The complexity of the low temperature MCD spectra of the oxidized and reduced trinuclear cluster shows the multiplicity of the predominantly S — Fe charge transfer transitions that contribute to the absorption envelope. While MCD spectroscopy provides a method of resolving the electronic transitions, assignment cannot be attempted without detailed knowledge of the electronic structure. However, the complexity of the low temperature MCD spectra is useful in that it furnishes a discriminating method for determining the type and redox state of protein bound iron-sulfur clusters. Each well characterized type of iron-sulfur cluster, i.e. [2Fe-2S], [3Fe-4S], and [4Fe-4S], has been shown to have a characteristic low temperature MCD spectrum in each paramagnetic redox state (1)... 

The technique of X-ray crystallography has been, and will remain, indispensable for the determination of the unusual structures of S—N compounds. A more recent development is the application of N NMR spectroscopy in S—N chemistry. Despite the necessity to employ N-enriched materials for these studies, the judicious application of this technique in both structural determinations and in monitoring the progress of reactions will undoubtedly accelerate the progress of the subject. The advent of MCD spectroscopy and the use of the perimeter model have also enhanced our understanding of the electronic structures of cyclic S—N molecules. Rapid advances in this area are to be expected. 

With the advances in experimental techniques and theory, MCD spectroscopy became a viable analytical technique and found application in many areas of chemistry (11-17). The distinct spectral forms of A, B, and C terms and the fact that MCD intensity can be positive or negative make an MCD measurement a useful complement to an absorption spectrum. MCD has been found to be rather more useful than MOR as the latter type of spectrum is generally more difficult to interpret (12). 

It was quickly recognized that a nonrelativistic formulation of MCD led to predictions that were qualitatively wrong for some paramagnetic metal halides (18-20). The introduction of spin-orbit coupling created addition contributions to the C term parameters and produced predictions in qualitative agreement with experiment. It was not until some time later that general formulations of the new terms caused by spin-orbit coupling appeared (21-23). The spin-orbit-induced C terms have played an important role in modem applications of MCD spectroscopy as they have proven to be extremely useful in the study of metalloen-zymes (15,24-26). 

Theoretical analysis has always been an important part of MCD spectroscopy. The parameters Aj, Bj, and Cj can be extracted from an experimental spectrum by a fit to a suitable set of functions or through the method of moments (27-28). The interpretation of these parameters is generally not a trivial task. For smaller, symmetrical molecules group theory has been used to good effect to extract information from an MCD spectrum (11). In recent years, quantum chemical calculation has proven a very useful aid in the interpretation of the often-complicated spectra of larger, nonsymmetric molecules. 

It was noted earlier that, in the absence of spin-orbit coupling, the spin part of the magnetic moment operator makes no contribution to MCD and spin degeneracy also plays no role in MCD spectroscopy. It was also noted that neglecting spin-orbit coupling led to qualitative errors in the predicted MCD spectra of some spin-degenerate systems. In this section we will present equations for the additional contributions to MCD that occur once spin-orbit coupling is included. 

The usefulness of MCD spectroscopy comes from the insight that it provides into the electronic structure of the ground and excited states of a molecule and into the nature of the transitions between these states. Much of this analysis comes from interpretation of the observed MCD intensity and measured MCD parameters in terms of the equations presented in Section II.A. In this section we will provide a few brief details about information that can be extracted from MCD parameters and some possible theoretical analysis techniques. 

Porphyrin complexes and the many important biological or commercial compounds that are based upon the porphyrin structure show distinct intense electronic spectra that vary in form depending on the exact structure of the ligand and the identity of the central metal ion. Simple porphyrin complexes have D4h symmetry as do many porphyrin derivatives. A large number of lower symmetry related molecules can be approximated successfully as D4h. Porphyrin complexes can be closed-shell or open-shell depending on the identity of the central metal. MCD spectroscopy, a technique that it sensitive to the space and spin symmetry of the ground... 

In recent years, some of the most impressive applications of MCD spectroscopy have come in the area of metalloenzymes... 

The absorption spectra of blue copper proteins typically include one major peak and two other peaks of varying size in the range 10,000-30,000 cm-1 (164-166). MCD spectroscopy has proved useful in assigning these peaks. The electronic excitations of the active site can be classed as either d—>d or LMCT transitions. The d- fd transitions will involve excited states where the electron hole remains on the Cu atom while the LMCT transitions will move the hole to the ligands, in particular the sulfur atoms of the Met and Cys groups. Thus the d- d transitions would be expected to be more strongly influenced by spin-orbit coupling and this should be reflected in the relative size of the Cj/Dj ratios of the bands in their MCD spectra. 

MCD spectroscopy has been useful in the location of the spin-forbidden transitions.1019 That the Cr—S bond is comparatively strong has been adduced from the presence of relatively intense molecular ion peaks in the mass spectra of several Cr111 dialkyldithiocarbamates.1020... 

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