Low-temperature MCD spectroscopy

Low-temperature MCD spectroscopy was used to probe the effects of binding the exogenous ligand azide to native laccase (89, 90). Titration of the native enzyme with azide produces two N3 - Cu(II) charge-transfer transitions one at 500 nm and a second more intense band at... 

Low-temperature MCD spectroscopy has been used to characterize the metal clusters present in the VFe protein of A. vinelandii (42). The temperature dependence of the MCD transitions of dithionite-reduced protein is similar to those arising from the S = 3/2 spin system of FeMoco of the MoFe proteins. The spectra of the VFe proteins have slightly different electronic and magnetic properties but provide strong spectroscopic evidence for the presence of a V-Fe-S cluster. In this oxidation state, only small additional spectral contributions from the two other paramagnetic species (S = 1/2 signal and the species detected in the dispersion mode) are observed. 

Trinuclear clusters have been detected in over 20 proteins as well as a number of enzymes, among them aconitase, beef heart succinate-ubiquinone oxidoreductase (120), Escherichia coli nitrate reductase (121), E. coli fumarate reductase (122), and succinate dehydrogenase (123). Selected instances of the occurrence of [3Fe-4S] clusters are listed in Table II. Because of the paramagnetic ground states of both oxidation levels, these clusters can be uniquely identified by a number of spectroscopic techniques. Among these, Mossbauer spectroscopy in applied magnetic fields (124, 128, 132, 141-143) and low temperature MCD spectroscopy (127, 138, 144-146) are decisive. While there are small spectroscopic differences among certain [3Fe-4S] centers, the similarities dominate and support the essential structure 3 for all. In a number of the earlier papers on protein... 

In complementary experiments, addition of H2O2 to the oxidized resting TlFIg Fc yielded a species that displayed the same EXAFS features as the peroxide-level intermediate. In this derivative, however, as opposed to the peroxide level intermediate, the T2 copper site is oxidized, allowing the use of EPR and low-temperature MCD spectroscopies to probe the coordination of the site. The data thus obtained, integrated with CD data (probing the T3 site), point to the formation of a peroxide bridge between the T2 site and one of the T3 coppers. 

Lehnert N (2008) Chapter 6 - EPR and low-temperature MCD spectroscopy of ferrous heme nitrosyls. In Abhik G (ed) The smallest biomolecules diatomics and their interactions with heme proteins. Elsevier, Amsterdam... 

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 low-temperature MCD and absorption titration studies (Figure 10) have determined that azide binds to both the type 2 and type 3 centers with similar binding constants. A series of chemical perturbations and stoichiometry studies have shown that these effects are associated with the same azide. This demonstrates that one N3 bridges between the type 2 and type 3 centers in laccase. These and other results from MCD spectroscopy first defined the presence of a trinuclear copper cluster active site in biology (89). At higher azide concentration, a second azide binds to the trinuclear site in laccase. Messerschmidt et al. have determined from X-ray crystallography that a trinuclear copper cluster site is also present in ascorbate oxidase (87, 92) and have obtained a crystal structure for a two-azide-bound derivative (87). It appears that some differences exist between the two-azide-bound laccase and ascorbate oxidase derivatives, and it will be important to spectroscopically correlate between these sites. 

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). 

Optical absorption spectra and MCD show the mixture of low-spin and high-spin states in cryoreduced HRP38 and HRP in complexes with F-, N3-, and CN-.40 Cryoreduced Fe3 + -NO complex of HRP was characterized by EPR and optical absorption spectroscopy.70 In all cases, the low-temperature primary products of cryogenic reduction relaxed to the equilibrium states of corresponding heme complexes. 

It is clear that a good deal of information needs to be obtained in order to interpret the structural data in particular, the question has to be faced as to whether the oxidised crystal structure is that of a resting state or whether the heme iron ligand switching occurs on each catalytic cycle. We do, however, know that the oxidised protein in solution has the same His/His coordination of the c-type heme as in the oxidised state of the crystalline enzyme. This was determined from MCD spectroscopy (Cheesman et al., 1997). Other solution spectroscocpic measurements have shown that the ligation of the d heme is very likely the same as in the crystalline state (Cheesman et al., 1997). These studies also showed that the d heme iron appeared to be in an unusual room temperature high/low spin equilibrium. 

Electronic spectroscopy (see Electronic Spectroscopy), in one form or another, has been the principal method used for the detection of short-lived intermediates. UV-visible absorption was the initial spectroscopic method used with flash photolysis and flow systems, and for each of these methods it remains the most commonly used approach. For species in low-temperature matrices, many varieties of electronic spectroscopy have been used. These include UV-visible absorption and emission, fluorescence, magnetic circular dichroism (MCD) and magnetic linear dichroism, and photoelectron spectroscopy. It is unfortunate, therefore, that in many cases electronic spectroscopy yields little or no stractnral information. The exceptions are high-resolution spectra, where vibrational or rotational flne structure may be seen. 

None directly the MHQ technique yields a frozen powder, which can be analyzed by various types of low-temperature spectroscopy like X-, Q-band EPR, UV-Visible spectroscopy, resonance Raman and potentially or in the near future by MCD, Mossbauer, ESEEM, ENDOR, EXAFS, W-, D-band EPR, MAS-NMR and FTIR spectroscopy. 

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