It has been shown by epr spectroscopy that at low

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It has been shown by EPR spectroscopy that at low temperature two cyanide anions bind to copper. The donor atoms are two cyanide carbon and two histidine nitrogen atoms in the basal plane, and the third histidine nitrogen in the axial position. 85 The hyperfine splitting is observed only with nuclei in the basal plane. It is observed both with I3C nuclei of I3C-enriched CN - and with the two 14N of two histidines. The second cya- nide may thus displace the coordinated water (Figure 2.17). Oxalate and sulfon- amides displace water from the coordination sphere. 85 ,86 For the oxalate ion this may occur through bidentate behavior. Coordination to an oxygen of the sulfon-
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70 2 I THE REACTION PATHWAYS OF ZINC ENZYMES AND RELATED BIOLOGICAL CATALYSTS amide cannot be ruled out, although the electronic and EPR spectra of the sul- fonamide complex are more consistent with a pseudotetrahedral chromophore. The S02 moiety would in any case point toward the B binding site. It is likely that sulfonamides bind as in ZnCA. Bicarbonate also shows less water relaxivity than other monodentate anions. 83,84.86 13C NMR spectroscopy has been used to investigate the location of the two substrates, CO 2 and HC0 3 ,with respect to the metal ion in CuCA. 8 6-88 As was pointed out in Section IV.B, the interconversion between the two species is slow on the NMR timescale in the absence of catalysts. Therefore, two sig- nals are observed (Figures 2.6 and 2.18). In the presence of the catalytically active CoCA, only one signal is observed at suitable enzyme concentrations, and individual information on CO 2 binding cannot be obtained. 89 ,90 In the pres- ence of inactive CuCA, two signals are again observed, which are broadened to different extents. For the HC0 3 - signal the Ti I values as estimated from the linewidth are much larger than TIl. Since the equation for Til, analogous to Equation (2.14), would predict similar T l and T 2 values,69,72 a sizeable broadening due to chem- ical exchange must be present. Indeed, unlike TiP I (Equation 2.1l), Tip I may be a complicated function of the exchange time TM and of the isotropic shift, ~WM, T- l _ fM TiJ + TiJTM: l + (~WM)2 2p - TM (TiJ + TM 1)2 + (~~)2 . (2.15) In the slow-exchange region, i.e., when two separate signals are observed and the broadening is due to exchange, Tip I = fMTM: 1. This region is characterized no enzyme A CoBCA II Figure 2.18 Schematic representation of the l3C NMR spectra of the C0 2 /HC0 3 - system (A) in pure water, (B) in the presence of CoCA, and (C) in the presence of CuCA. 56 162 1\ CuBCA II is (ppm) B C
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IV. ELUCIDATION OF STRUCTURE-FUNCTION RELATIONSHIPS: CARBONIC ANHYDRASE 71 by a marked increase in linewidth with increasing temperature, as confirmed by measurements at 4 and 25°C. Therefore, T 2p gives a direct measure of 'TM. 56 The 13C TIl values of HC0 3 - are consistent with bicarbonate bound to the metal.
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