A

Time, fis

2 4 6 8 Frequency, MHz

Fig. 6. ESEEM spectra for T2D Fet3p (A) the ESEEM spectrum of the type-1 Cu(II) site (T2D mutant) was collected at 2825 G with t = 250 ns. The measurement conditions were 8.80 GHz microwave frequency, 43 dB microwave power, and 4.2 K sample temperature. The sample was 0.5 mM Fet3p in pH = 6.0 MES buffer containing 25% ethylene glycol. (B) Fourier transform of the ESEEM data. (From ref. 14.)

three-coordinate site with a significant doming of the Cu(I) out of the trigonal plane, presumably toward a more weakly bound fourth ligand.

The EXAFS analysis of the reduced and the oxidized T1D/T2D mutant protein and of the T1D mutant that had an intact trinuclear center gave a rather complete picture of the coordination at the type-3 cluster. The best fit of the EXAFS data in all cases included the coordination of three histidine imidazoles to each of the two type-3 coppers consistent with the K-edge results. Furthermore, the bridging oxygen ligand that electronically couples the two Cu(II) in the oxidized state was strongly evident in the data. Also evident was the scattering that gave a Cu-Cu distance in the cluster of 3.33 A. The fitted data for the oxidized cluster are indicated in the structure shown in Fig. 12A.

The analysis of the data for the reduced protein was slightly more ambiguous. This ambiguity followed from the fact that the best fit required an additional single oxygen or nitrogen first shell atom at 2.49 A distributed between the two copper atoms. This was at a considerably longer distance than the bridging O in the oxidized cluster (2.49 vs 1.91 A; see Fig. 12A,B). Also, the Cu-Cu distance in the reduced cluster was greater because there was no evidence of Cu backscattering in the data. One interpretation of these data is given in Fig. 12B. In the model, the two copper atoms move apart upon reduction. The oxygen bridge is disrupted and the putative hydroxide is protonated, thus making cluster reduction electroneutral. (As presented in Fig. 12, the formal charge of both the oxidized and reduced states is +2; this is arbitrary, of course, because it depends on how one writes the

Fig. 7. ESEEM spectra for T1D Fet3p. (A) ESEEM spectrum of the type-2 Cu(II) site was collected at 2690 G with t = 260 ns. The measurement conditions were 8.80 GHz microwave frequency, 43 dB microwave power, and 4.2 K sample temperature. The sample was 0.5 mM Fet3p in pH = 6.0 MES buffer containing 25% ethylene glycol. (B) Fourier transform of the ESEEM data. (From ref. 14.)

0 0

Post a comment