Protein complex formation is thought to be at least a two-step process, in which the active complex is preceded by the formation of an encounter complex. The interactions in the encounter complex are usually dominated by electrostatic forces, whereas the active complex is also stabilized by noncovalent short-range forces. Here, the complex of cytochrome f and plastocyanin, electron-transfer proteins involved in photosynthesis, was studied using paramagnetic relaxation NMR spectroscopy. Spin labels were attached to cytochrome f, and the relaxation enhancements of plastocyanin nuclei were measured, demonstrating that a large part of the cytochrome f surface area is sampled by plastocyanin. In contrast, plastocyanin is always oriented with its hydrophobic patch toward cytochrome f. The complex was visualized using ensemble docking, showing that the encounter complex is stabilized by hydrophobic as well as electrostatic interactions. The results suggest a model of electrostatic preorientation before the proteins make contact, followed by the formation of an encounter complex that rapidly leads to electron-transfer active conformations by gradual increase of the overlap of nonpolar surface areas on cytochrome f and plastocyanin. In this model the distinction between the encounter and active complexes vanishes, at least in the case of electron-transfer complexes, which do not require a high degree of specificity.

Electrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redoxp roteins is often optimizedt os teer ar edox partner to the electron transfer active binding site.T ot est whether the optimizeddistribution is more important than the strength of the electrostatic interactions,anadditional negative patch was introduced on the surface of cytochrome cp eroxidase,a way from the stereospecific binding site,a nd its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner,c ytochrome c, interacts with the new patch.U nexpectedly,t he rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimizedc harge distribution.

Electrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redoxp roteins is often optimizedt os teer ar edox partner to the electron transfer active binding site.T ot est whether the optimizeddistribution is more important than the strength of the electrostatic interactions,anadditional negative patch was introduced on the surface of cytochrome cp eroxidase,a way from the stereospecific binding site,a nd its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner,c ytochrome c, interacts with the new patch.U nexpectedly,t he rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimizedc harge distribution.

Protein complex formation depends strongly on electrostatic interactions. The distribution of charges on the surface of redox proteins is often optimized by evolution to guide recognition and binding. To test the degree to which the electrostatic interactions between cytochrome c peroxidase (CcP) and cytochrome c (Cc) are optimized, we produced five CcP variants, each with a different charge distribution on the surface. Monte Carlo simulations show that the addition of negative charges attracts Cc to the new patches, and the neutralization of the charges in the regular, stereospecific binding site for Cc abolishes the electrostatic interactions in that region entirely. For CcP variants with the charges in the regular binding site intact, additional negative patches slightly enhance productive complex formation, despite disrupting the optimized charge distribution. Removal of the charges in the regular binding site results in a dramatic decrease in the complex formation rate, even in the presence of highly negative patches elsewhere on the surface. We conclude that additional charge patches can result in either productive or futile encounter complexes, depending on whether negative residues are located also in the regular binding site.

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b6f with the electron transfer proteins plastocyanin and cytochrome c6. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c6 from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c6 were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge-charge interactions pre-orient cytochrome c6 with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.