The spectra in 2 for both kinds of derivatives show

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sharper signals than four-coordinate ones. The spectra in 0 2 0 for both kinds of derivatives show three fewer isotropically shifted signals than in H 2 0. These signals are assigned to histidine NH protons, which are replaced by deuterons in 0 2 0. Five-coordinate species provide IH NMR spectra with many signals slightly shifted from the diamagnetic position. It is believed that such complexes have relatively large magnetic anisotropy, which, summed up to the external magnetic field, provides further differentiation in shifts of the protons. Such shift contributions are called pseudocontact shifts. These shifts depend on the third power of the distance from the metal and on the position of the proton
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65 CoCA pH 10.0 CoCA.acetazolamide pH 8.5 H ~~~ 160 140 120 100 80 60 40 20 0 ·20 ·40 ·60 ·80 ·100 ·120 ppm CoCA.I- pH 6.5 5.6 7.6'----,(1, __ CoCA.NCS- pH 6.7 CoCA. NO, pH 6.0 CocA.c,oi- pH 6.5 CoCA.Au(CN), pH 6.3 4.3 160 140 120 100 80 60 40 20 0 ·20 ·40 ·60 ·80 ·100 ·120 ppm Figure 2.14 IH NMR spectra of cobalt(II)-substituted bovine carbonic anhydrase II and some inhibitor deriv- atives. The three sharp downfield signals in each spectrum disappear in D 2 0 and are assigned to the exchangeable ring NH protons of the three coordinated histidines. The sharp signal labeled with an arrow is assigned to the H8Z proton of His-119, which is the only non-exchangeable ring proton in a meta-like rather than in an ortho-like position with respect to the coordinating nitrogen. The T 1 values (ms) of the signals for the I - and C Z 0 4 2 - derivatives are also shown. 25 ,75 with respect to the molecular axes. These signals belong to protons of noncoor- dinated residues from 5 to 10 A from the metal. Their assignment in principle provides further information on the structure in the vicinity of the metal ion. The 1 H NMR spectra of cobalt(II) enzymes thus afford a powerful method for monitoring structure and reactivity of the metal-bound residues. This is one task for future investigations of the enzyme. 4. pH dependence of inhibitor binding The ease with which electronic spectra can be obtained provides a simple way of determining the affinity constants of inhibitors for the cobalt-substituted enzymes. An aliquot of enzyme is diluted in a spectrophotometric cell up to a
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66 HeN sulfonamides imidazole triazoles pKalEJ pH Figure 2.15 Types of pH dependences observed for the affinity constants of inhibitors for cobalt(II)-substituted carbonic anhydrases. pKalE] represents the main pK a value of the enzyme, pKalI] that of the inhibitor, if present. 48 fixed volume, and the spectrum is measured. Then the spectra are remeasured on samples containing the same amount of enzyme plus increasing amounts of inhibitor in the same cell volume. The pH is rigorously controlled. If solutions of enzyme and inhibitor have the same pH, the pH should be verified after the spectral measurements, in order to avoid contamination from the electrode salt medium. Both absolute values and pH dependences of affinity constants ob- tained from electronic spectra are the same as those obtained from inhibition
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