Protein Docking and Gated Electron-Transfer Reactions between Zinc Cytochrome c and the New Plastocyanin from the Fern Dryopteris crassirhizoma. Direct Kinetic Evidence for Multiple Bina
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- 作者：Ekaterina V. Pletneva ; D. Bruce Fulton ; Takamitsu Kohzuma ; and Nenad M. Kosti
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- 刊名：Journal of the American Chemical Society
- 出版年：2000
- 出版时间：February 16, 2000
- 年：2000
- 卷：122
- 期：6
- 页码：1034 - 1046
- 全文大小：430K
- 年卷期：v.122,no.6(February 16, 2000)
- ISSN：1520-5126
文摘
A new plastocyanin from the fern Dryopteris crassirhizoma markedly differs from other plastocyaninsin having a very large acidic surface, which extends into the area that is hydrophobic in other plastocyanins.The exceptionally large dipole moment of 439 D has a completely different orientation and protrudes throughthe "northwest" region of the surface, which is now acidic. Consequently, the new plastocyanin differs fromits congeners in the photoinduced reaction with zinc cytochrome c: 3Zncyt + pc(II) 
Zncyt+ + pc(I). Ationic strength 
20 mM and solution viscosity 1.8 cp, at least three exponentials are needed to describe theoxidative quenching of 3Zncyt. Besides a bimolecular phase, there are two distinct unimolecular phasescorresponding to electron transfer within two different persistent complexes 3Zncyt/pc(II). So-called normaland reverse titrations yield consistent values of the unimolecular rate constants: k1 is (3.3 ± 0.7) × 105 s-1and (3.2 ± 0.4) × 105 s-1, and k2 is (7.6 ± 0.8) × 103 s-1 and (8.2 ± 1.2) × 103 s-1. The respective 
H
values also differ (16 ± 2 and 27 ± 7 kJ/mol), but S values are the same (-88 ± 7 and -78 ± 23 J/K mol).Viscosity effects and also unrealistic reorganizational energies obtained in fittings of temperature effects toMarcus theory reveal that both unimolecular electron-transfer reactions (k1 and k2) are gated by structuralrearrangement of the respective binary complexes. Additional evidence for multiple persistent binary complexesis dependence on ionic strength of the apparent rate constant kapp for electron transfer in the transient binarycomplex 3Zncyt/pc(II). Analysis of this dependence indicates that rearrangement of the protein complexesinvolves relatively large migration of zinc cytochrome c, which is facilitated at higher ionic strength. Whenzinc cytochrome c is present in excess, a transient, but not persistent, ternary complex Zncyt/pc/Zncyt is formed;both reverse titration and analysis of the effects of protein association on the 1H NMR chemical shifts supportthis conclusion. Existence of a ternary complex is consistent with the existence of multiple binary complexes.Monte Carlo simulations show possible docking configurations of the binary Zncyt/pc complexes. Thesetheoretical calculations, in conjunction with our kinetic data, suggest that the faster (k1) and slower (k2)intracomplex reactions seem to occur when 3Zncyt docks, respectively, in the "northeast" and "northwest"surface regions of fern plastocyanin (in the conventional orientation). The new type of docking, on the"northwest" side of the plastocyanin surface, is favored by new acidic residues in this region.
Zncyt+ + pc(I). Ationic strength 20 mM and solution viscosity 1.8 cp, at least three exponentials are needed to describe theoxidative quenching of 3Zncyt. Besides a bimolecular phase, there are two distinct unimolecular phasescorresponding to electron transfer within two different persistent complexes 3Zncyt/pc(II). So-called normaland reverse titrations yield consistent values of the unimolecular rate constants: k1 is (3.3 ± 0.7) × 105 s-1and (3.2 ± 0.4) × 105 s-1, and k2 is (7.6 ± 0.8) × 103 s-1 and (8.2 ± 1.2) × 103 s-1. The respective Hvalues also differ (16 ± 2 and 27 ± 7 kJ/mol), but S values are the same (-88 ± 7 and -78 ± 23 J/K mol).Viscosity effects and also unrealistic reorganizational energies obtained in fittings of temperature effects toMarcus theory reveal that both unimolecular electron-transfer reactions (k1 and k2) are gated by structuralrearrangement of the respective binary complexes. Additional evidence for multiple persistent binary complexesis dependence on ionic strength of the apparent rate constant kapp for electron transfer in the transient binarycomplex 3Zncyt/pc(II). Analysis of this dependence indicates that rearrangement of the protein complexesinvolves relatively large migration of zinc cytochrome c, which is facilitated at higher ionic strength. Whenzinc cytochrome c is present in excess, a transient, but not persistent, ternary complex Zncyt/pc/Zncyt is formed;both reverse titration and analysis of the effects of protein association on the 1H NMR chemical shifts supportthis conclusion. Existence of a ternary complex is consistent with the existence of multiple binary complexes.Monte Carlo simulations show possible docking configurations of the binary Zncyt/pc complexes. Thesetheoretical calculations, in conjunction with our kinetic data, suggest that the faster (k1) and slower (k2)intracomplex reactions seem to occur when 3Zncyt docks, respectively, in the "northeast" and "northwest"surface regions of fern plastocyanin (in the conventional orientation). The new type of docking, on the"northwest" side of the plastocyanin surface, is favored by new acidic residues in this region.