Native Paramagnetic Centers

If the metal has zero total electron spin, a complex is EPR active only if one of the ligands has an odd number of electrons, important examples being NO (see Section 5), NO2 [15], and semiquinone radicals [16]. 

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The FeMo cofactor (or M center) in the MoFe-proteinis in the native paramagnetic M state. Reduction of the MoFe-protein by the Fe-protein results in the reduction of FeMo-co from the M state to the M state at a potential estimated to be less than —0.465 V (NHE). The electron paramagnetic resonance (EPR) silent M state is only transiently produced during catalysis, and relaxes to the M state when catalysis stops. The intimate consequences of the M state reduction are not precisely known. A more oxidized diamagnetic state may also be generated (M ) at —0.042 V but its biological relevance is unclear [9]. 

Cua centers exist in two redox states [Cu(II)Cu(I)] and [Cu(I)Cu(I)]. The oxidized species is a fully delocalized mixed-valence pair (formally two Cu+ 1.5 ions), as revealed by EPR spectroscopy (Kroneck et al., 1988, 1990). Despite the similar coordination geometry around copper, these systems display sharper NMR lines than do the BCP due to a shorter electron relaxation time of the paramagnetic center (wlO "s) (dementi and Luchinat, 1998). NMR studies are available for the native Cua centers from the soluble fragments of the The. thermophilus, Paracoc-cus denitrificans, Paracoccus versutus, and Bacillus subtilis oxidases (Bertini et al., 1996 Dennison et al., 1995 Luchinat et al., 1997 Salgado et al., 1998a) and Pseudomonas stutzeri N2O reductase (Holz et al., 1999), as well as for engineered Cua sites in amicyanin (Dennison et al., 1997) and Escherichia coli quinol oxidase (Kolczak et al., 1999). 

For the type 3 center, the antiferromagnetic coupling leads to an S = 0 ground state that cannot split in a magnetic field. Thus, this site does not exhibit C-term intensity and the low-temperature MCD spectrum of native laccase will be dominated by the intense C-terms associated with paramagnetic copper centers (89, 90). 

An x-ray study of the cyanate and cyanide derivatives of the native enzyme has shown that the anions sit in the cavity without binding to the metal ion (M. Lindahl, L.A. Svensson, and A. Liljas, Proteins 15 (1993), 177). Since NCO has been shown to interact with the paramagnetic cobalt(II) center, and C-enriched cyanide has been shown to interact with Zn-substituted CA (see Reference 67), it appears that the structures in the solid state and solution are strikingly different. 

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