Quantitative Analysis Of Thermodynamic And Kinetic Properties Of The Electrontransfer Process Mediated By Spinach Plastocyanin

We now shift focus from the classification of the different cupredoxin subfamilies to the study of a particular blue copper protein, the plastocyanin from spinach. This protein is selected as it has been subjected to extensive experimental characterization; a similar computational analysis could be applied to other Cu proteins.

Plastocyanins consist of approx 100 amino acid residues (4). They are found in the chloroplasts of higher plants, blue-green algae, and photosynthetic cyanobacteria, where they take part in the photo-synthetic process by transporting electrons from the membrane-bound heme protein cytochrome-f (cyt f) to the chlorophyll-containing pigment P700 of Photosystem I (PSI).

Photosynthesis allows organisms to transform light energy into the chemical energy they need to survive, through a complex series of interrelated molecular processes and physicochemical reactions. Discovering the secrets of the photosynthetic process is an intriguing and important challenge for the

Fig. 8. Three-dimensional structure of spinach plastocyanin, the amino acids constituting the small and large acidic patch at the eastern site of the protein are highlighted.

research community, especially in view of the fact that it can provide virtually all of the energy for all living organisms on our planet to survive.

The plastocyanins are the best studied and structurally characterized cupredoxins. In addition, many experimental thermodynamic and kinetic studies have been performed on the overall electron-transfer reactions between plastocyanin and its electron donor, cytochrome-f. All of the data collected (5,6,9,11,25) indicate that the overall reaction between plastocyanin and cytochrome-f, k2

can be described, to a first approximation, as a three-step process:

■ (PC0X/CYTred)a ~ (PC0X/CYTred)b ~ (PCred/CYT0X) ~ PCred + CYT0

where kon and koff are respectively the on and off rate constants for association of the oxidized plastocyanin (PCox) and reduced cytochrome-f (CYTred), which might be different from those (k' on and k' off) for dissociation of the PCred/CYTox complex; krearr and kder are the forward and reverse rate constants, respectively, for the rearrangement of the complex; and ket and kret are the forward and reverse electron-transfer rate constants, respectively.

Two different regions on the plastocyanin surface are thought to be involved in different steps of the overall reaction: the so-called "eastern" site and "northern" site, which are highlighted in Fig. 1.

The eastern site is the large negatively charged surface area that is a distinctive feature of the higher plant plastocyanins and consists of two negatively charged patches (the "acidic patches") that surround the solvent-exposed Tyr83. The large acidic patch includes residues Asp42, Glu43, Asp44, and Glu45, and the small acidic patch includes residues Glu59, Glu60 and Asp61 (Figs. 1 and 8). Evidence

PCox + CYT

Fig. 9. Three-dimensional structure of spinach plastocyanin with the aminoacids mutated at the northern site highlighted.

has been found for this site to be involved in the association process of plastocyanin and cytochrome-f (4). The hypothesis that the eukaryotic plastocyanin eastern site is likely to be necessary for recognition of the basic patch in cytochrome-f and assembling of an electrostatic complex is supported by the NMR-based structures of the spinach plastocyanin/turnip cytochrome-f complex (21,22).

The northern site is the hydrophobic region that contains the Cu and is, as discussed previously, almost conserved in all of the cupredoxin subfamilies and therefore commonly thought to be involved in the electron-transfer process. The electron-transfer properties of plastocyanin are largely determined by its redox potential and thermodynamic parameters for the oxidation/reduction of Cu(I)/ Cu(II). In fact, the reduction potential (E°) determines the ability of the redox protein to oxidize its electron-donor partner and reduce its electron-acceptor partner, therefore influencing the overall process of electron transfer mediated by the protein. Variations of the reduction potential and the ther-modynamic properties of the copper center are interesting factors to be analyzed, especially in view of the fact that they depend, in addition to Cu-ligand interactions, on the charge distribution of the protein surface and on the electrostatic properties and ionic strength of the environment, which are all factors also influencing the recognition and association events prior to electron transfer (Eq. 9). For example, the perturbation of the surface charge distribution due to mutations can, on one hand, influence the ability of the protein to attract its partner, thus influencing their association, and, on the other hand, modify the hydrogen-bonding network to varying extents in the two redox states, thus promoting a selective stabilisation of one with respect to the other and affecting the redox properties.

Here, two different subsets of plastocyanin mutants, located in the northern and in the eastern site regions, are analyzed. For this study, the plastocyanin from spinach was chosen, because the structure of this plastocyanin variant is known both in its isolated form and in a complex with a cyto-chrome-f from turnip (21,22,37). The aim is to investigate whether and to what extent it is possible to rationalize variations in kinetic properties and redox thermodynamic parameters observed experi mentally for the spinach plastocyanin on the basis of structural and electrostatic modifications of the protein. This is done by applying a QSPR/QSAR approach, where descriptors of different types were tested. The technique used allows the prediction of protein functionality and the design of new protein variants (in particular, mutant proteins) with desired activities.

3.1. QSPR Analysis: The Structural Molecular Determinants of the Spinach Plastocyanin Reduction Thermodynamics

A debated issue in biological redox chemistry is how the two oxidation states of the copper atom in cupredoxins [Cu(I)/Cu(II)] are stabilized by the protein matrix and the solvent and, consequently, which relationship can be established between the observed reduction potential (E°' ) and the protein structure/sequence features. Understanding the interconnection of these effects would allow, on one hand, the prediction of the functional properties of cupredoxin mutants and, on the other hand, the engineering of new proteins with desired redox activity and thermodynamic properties.

With this aim, it is particularly interesting to study the effects of point mutations altering the electrostatic potential in the region of the copper on the thermodynamics of Cu(II) reduction. The variation in the reduction enthalpy and entropy is analyzed by a QSPR approach, in order to derive quantitative models for the rationalization and interpretation of the observed behaviors (38).

The functionally important residues Leu12 and Gln88 are replaced with charged and polar residues (L12E, L12K, L12H, L12Q, L12G, Q88E, and Q88K) and Asn38 is substituted with Asp (N38D). The location of these three residues is highlighted in Fig. 9. Both Leu12 and Gln88 are surface residues. The former contributes to the hydrophobic patch at the northern site of plastocyanin and seems to be involved in the interaction with photosystem I (PSI) (39), whereas the latter protrudes through the acidic eastern site of the protein, which is involved in the interaction with cyto-chrome-f (21). Although Asn38 does not contribute to the protein surface, it has been shown to occupy a strategic position for the stabilization of the architecture of the copper site (40). In addition, both Gln88 and Asn38 are adjacent to a Cu-ligand histidine: His 87 and His37, respectively. Therefore, investigating whether mutations of these residues might alter the structure of the copper coordination region and, as a consequence, its functional properties would be a first step toward comprehension of the structural mechanisms that regulate the protein redox capabilities.

3.1.1. Selected Descriptors

The experimental data to be rationalized are obtained with cyclic voltammetry and relate to the Cu(II) to Cu(I) reduction thermodynamics of plastocyanin mutants (38). The entropic (A5°') and enthalpic (AH°') contributions to the redox potential E°' are reported in Table 4, together with the values of the most representative computed descriptors. All descriptors are computed on the minimized molecular dynamics average structure of the proteins, as described in detail in ref. 38. Among the many computed descriptor, the most interesting and explanatory descriptors are as follows:

1. Dz. The component of the dipole moment of the proteins along the z axis, which has its origin at the center of mass of the protein and points toward the copper site. This property was computed with the UHBD program (32).

2. BIC. The bonding information content defined by Shannon information theory (41). According to its definition, the BIC index encodes the branching ratio, unsaturation, and constitutional diversity of a molecule.

3. SI. The similarity index computed by comparing, in a pairwise fashion, the magnitude and the distributions of MEPs of the wt plastocyanin and its mutants. This comparison is performed in a restricted region close to the protein "northern" site.

4. DPSA. The difference in the charged partial surface areas of the proteins (42), which are computed on the minimized average structures: DPSA = PPSA - PNSA, where PPSA is the partial positive [(^(+SA;)] and PNSA is the partial negative [¿'(-SA,)] surface area of the proteins. +SA; and -SA; are the surface area contributions of the ith positive and negative atom, respectively.

Table 4

Experimental Thermodynamic Parameters and Theoretical Molecular Descriptors Computed on the Minimized Average Structure of Plasocyanin and Its Mutants and Used for QSAR Modeling

Table 4

Experimental Thermodynamic Parameters and Theoretical Molecular Descriptors Computed on the Minimized Average Structure of Plasocyanin and Its Mutants and Used for QSAR Modeling

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