Vi

Fig. 11. Correlations between experimental thermodynamic parameters AH°'rc (A), ASs'rc (B and C), E°' (D), and protein structure descriptors.

amount of dispersion interactions within the protein. The variation in the reduction entropy resulting from mutations appears to be linked to the hydrogen-bonding donor/acceptor character of the northern part of the protein, above the metal site, and to the perturbation of the structure and the electrostatic potential distribution around the copper site with respect to the wild-type plastocyanin. These properties influence the reduction-induced reorganization of the water molecules on the protein surface in the same region.

3.2. QSAR Analysis: The Structural Molecular Determinants of the Spinach Plastocyanin/Cytochrome-f Association Kinetics

The main goal is to establish relationships between structural and kinetic parameters determined for the overall electron-transfer process between cytchrome-f and plastocyanin [Eq. (8)]. In particular, we will investigate the role of the negative patch on the eukaryotic plastocyanin eastern site in the recognition process, first step of Eq. (9), and, as a consequence, in the overall electron-transfer processes with cytochrome-f. The availability of experimental values of the overall kinetic constants k2 [Eq. (8)], which were determined by Kannt et al. (24) for the in vitro electron-transfer reaction between wild-type and mutant spinach plastocyanins (D42N, E43N, E43K, E43Q/D44N, E59K/E60Q, E59K/E60Q/E43N, and Q88E) and the soluble part of turnip cytochrome-f, allows comparisons and QSAR analyses.

The relevance of the negative patch for the interaction between pc and cyt f is highlighted by the structure of the complex obtained from NMR data, which is shown in Fig. 12. The mutated residues in the eastern site (D42N, E43N, E43K, E43Q/D44N, E59K/E60Q, E59K/E60Q/E43N, and Q88E) are shown in Fig. 8. Residues D42, E43, and D44 are in the large acidic patch, whereas residues E59 and E60 are in the small acidic patch. Residue Q88 is not part of the acidic area, although it makes interactions with Y83, which lies almost in the middle of the eastern site and is thought to be a possible entry/exit point for electrons. Furthermore, as previously stated, Q88 is covalently bound to the Cu ligand H87.

The experimental data available from Kannt et al. (24) relative to mutations in the eastern site are collected in Table 5, together with the indices used for QSAR analysis.

3.2.1. Selected Descriptors

The X-ray structure deposited in the pdb (1ag6) was used for the QSAR analysis. The protein was mutated (D8G) to the spinach plastocyanin wt sequence (according to the SWISSPROT protein sequence database). Afterward, mutants (D42N, E43K, E43N, Q88E, E43Q/D44N, E59K/E60Q, E59K/E60Q/E43N) were produced by substituting the appropriate residues.

The molecular electrostatic potentials of the proteins were computed with the UHBD program (32) in a way similar to that described in Section 2.2. The Hodgkin similarity index [SI, Eq. (5)] (33), computed between the molecular electrostatic potential of each mutant and the wt protein, and the derived index sqrt(2-2SI) were used as quantitative descriptors of the perturbation of the molecular electrostatic potential of wt plastocyanin caused by the introduction of mutations at the eastern site of the protein. The SI provides a measure of the similarity between the magnitudes and distributions of the molecular electrostatic potentials of the considered mutant and the wild type. The sqrt(2-2SI) index is instead a measure of the difference only in the magnitude of the MEPs of the two proteins compared, assuming that these MEPs are not highly different. This assumption is reasonable because only a few residues (either one, two, or three) are mutated at the same time.

Assuming that all the wild-type and mutant forms under study interact with cytochrome-f with overall similar features, the SI and sqrt(2-2SI) values relate to the electrostatic enhancement of association rates of pc mutants with cyt f. Therefore, these indices can be employed in QSAR analyses, in correlation with the experimental overall rate constants to estimate whether and to what extent mutations in the eastern acidic area influence the overall electron-transfer reaction by affecting the association step and/or the electron-transfer step (Eq. 9).

Fig. 12. NMR-based 3D structure of the spinach plastocyanin/turnip cytochrome-f complex (21). The metal ion (which defines the Cu site or northern site) in the plastocyanin and the heme cofactor in the cytochrome-f structure are indicated. The eastern site in plastocyanin is also highlighted.

Eastern site

Fig. 12. NMR-based 3D structure of the spinach plastocyanin/turnip cytochrome-f complex (21). The metal ion (which defines the Cu site or northern site) in the plastocyanin and the heme cofactor in the cytochrome-f structure are indicated. The eastern site in plastocyanin is also highlighted.

Table 5

Experimental k2 Values for the Reaction of wt and Mutant Plastocyanins with Cytochrome-f (25) and ln(k2/k2 WT) Values at pH 7.5 and 6.0; SI Values Computed by Comparing the MEPs of the Mutants Relative to the wt Plastocyanin on the Complete Skin [SI and sqrt(2-2SI)] and on the Protein Eastern Site [Sles and sqrt (2-2SIes)]

Table 5

Experimental k2 Values for the Reaction of wt and Mutant Plastocyanins with Cytochrome-f (25) and ln(k2/k2 WT) Values at pH 7.5 and 6.0; SI Values Computed by Comparing the MEPs of the Mutants Relative to the wt Plastocyanin on the Complete Skin [SI and sqrt(2-2SI)] and on the Protein Eastern Site [Sles and sqrt (2-2SIes)]

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