Loss of Electrostatic Interactions Causes Increase of Dynamics within the Plastocyanin–Cytochrome f Complex

Abstract

Recent studies on the electron transfer complex formed by cytochrome f and plastocyanin from Nostoc revealed that both hydrophobic and electrostatic interactions play a role in the process of complex formation. To study the balance between these two types of interactions in the encounter and the final state, the complex between plastocyanin from Phormidium laminosum and cytochrome f from Nostoc sp. PCC 7119 was investigated using NMR spectroscopy and Monte Carlo docking. Cytochrome f has a highly negative charge. Phormidium plastocyanin is similar to that from Nostoc, but the net charge of the protein is negative rather than positive. NMR titrations of Zn-substituted Phormidium plastocyanin and Nostoc cytochrome f indicated that a complex with an affinity intermediate between those of the Nostoc and Phormidium complexes is formed. Plastocyanin was found in a head-on orientation, as determined using pseudocontact shifts, similar to that in the Phormidium complex, in which the hydrophobic patch represents the main site of interaction on plastocyanin. However, the interaction in the cross-complex is dependent on electrostatics, similar to that in the Nostoc complex. The negative charge of plastocyanin decreases, but not abolishes, the attraction to cytochrome f, resulting in the formation of a more diffuse encounter complex than in the Nostoc case, as could be determined using paramagnetic relaxation spectroscopy. This work illustrates the subtle interplay of electrostatic and hydrophobic interactions in the formation of transient protein complexes. The results are discussed in the context of a model for association on the basis of hydrophobic contacts in the encounter state.

Molecular Organization of Cytochrome c2 near the Binding Domain of Cytochrome bc1 Studied by Electron Spin–Lattice Relaxation Enhancement

“…The approach was based on measurements of magnetic interactions between spin label (SL) on cytochrome c 2 and a fast-relaxing iron ion of heme c 1 in cytochrome c 1 . These interactions were detected as paramagnetic relaxation enhancement (PRE), 18 a phenomenon that has been exploited to study protein–protein interactions by both EPR and NMR spectroscopy, in systems such as cytochrome c –cytochrome c oxidase, 19 cytochrome c –cytochrome c peroxidase, 20,21 plastocyanin–cytochrome f , 22 and Rieske protein–cytochrome b . 23 Our experiments further support the collisional mechanism in which cytochrome bc 1 does not form long-lived complexes with cytochrome c 2 and ET between the proteins is a product of several collisions between proteins.…”

Identification of productive and futile encounters in an electron transfer protein complex

“…In the encounter complex, the proteins assume multiple other orientations, often in equilibrium with the major stereospecific state. In low-affinity complexes with dissociation constant (K d ) values > 10 μM, the encounter complex can represent a sizeable fraction, and in some cases, a well-defined, stereospecific complex may even be absent (10)(11)(12)(13)(14)(15). The presence of encounter states may be a consequence of the chemical nature of proteins.…”

The Transient Complex of Cytochrome c and Cytochrome c Peroxidase: Insights into the Encounter Complex from Multifrequency EPR and NMR Spectroscopy

“…Previous experiments suggest that the initial stage of the formation of the encounter complex is governed mostly by long‐range electrostatic interactions, but hydrophobic interactions can in some cases be relevant . Guiding the proteins through these interactions, the encounter complex can enhance the formation of the stereo‐specific complex by reducing the search area.…”