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 binary complexes

A new plastocyanin from the fern Dryopteris crassirhizoma markedly differs from other plastocyanins in having a very large acidic surface, which exten ds into the area that is hydrophobic in other plastocyanins. The exceptiona lly large dipole moment of 439 D has a completely different orientation and protrudes through the "northwest" region of the surface, which is now acid ic. Consequently, the new plastocyanin differs from its congeners in the ph otoinduced reaction with zinc cytochrome c: (3)Zncyt + pc(II)--> Zncyt(+) pc(I). At ionic strength less than or equal to 20 mM and solution viscosit y (less than or equal to 1.8 cp, at least three exponentials are needed to describe the oxidative quenching of 3Zncyt. Besides a bimolecular phase, th en, are two distinct unimolecular phases corresponding to electron transfer within two different persistent complexes (3)Zncyt/pc(II). So-called norma l and reverse titrations yield consistent values of the unimolecular rate c onstants: k(1) is (3.3 +/- 0.7) x 10(5) s(-1) and (3.2 +/- 0.4) x 105 s(-1) , and kz is (7.6 +/- 0.8) x 10(3) s(-1) and (8.2 + 1.2) x 103 s(-1). The re spective Delta H-double dagger values also differ (16 +/- 2 and 27 +/- 7 kJ /mol), but Delta S-double dagger values are the same (-88 +/- 7 and -78 +/- 23 J/K mel). Viscosity effects and also unrealistic reorganizational energ ies obtained in fittings of temperature effects to Marcus theory reveal tha t both unimolecular electron-transfer reactions (k(1) and kz) are gated by structural rearrangement of the respective binary complexes. Additional evi dence for multiple persistent binary complexes is dependence on ionic stren gth of the apparent rate constant ii,,, for electron transfer in the transi ent binary complex 3Zncyt/pc(II). Analysis of this dependence indicates tha t rearrangement, of the: protein complexes involves relatively large migrat ion of zinc cytochrome c, which is facilitated at higher ionic strength. Wh en zinc cytochrome c is present in excess, a transient, but not persistent, ternary complex Zncyt/pc/Zncyt is formed; both reverse titration and analy sis of the effects of protein association on the H-1 NMR chemical shifts su pport this conclusion. Existence of a ternary complex is consistent with th e existence bf multiple binary complexes. Monte Carlo simulations show poss ible docking configurations of the binary Zncyt/pc complexes. These theoret ical calculations, in conjunction with our I;kinetic data, suggest that: th e faster (k(1)) and slower (kr) intracomplex reactions seem to occur when 3 Zncyt docks, respectively, in the "'northeast" and "northwest" surface regi ons of fern plastocyanin lin the conventional orientation). The new type of docking, on the "northwest" side of the plastocyanin surface, is favored b y new acidic residues in this region.