The complex of the photosynthetic redox partners plastocyanin and cytochrome f from the thermophilic cyanobacterium, Phormidium laminosum, was investigated by nuclear magnetic resonance (NMR). Chemical-shift perturbation analysis of amide proton and nitrogen nuclei implicates the hydrophobic patch and, to a lesser extent, the "eastern face" of plastocyanin in the complex interface. Intermolecular pseudocontact shifts observed in the complex of cadmium-substituted plastocyanin and ferric cytochrome f specifically define the site of interaction to be between the hydrophobic patch of plastocyanin and the heme region of cytochrome f. Rigid-body structure calculations using NMR-derived restraints demonstrate that plastocyanin is oriented in a "head-on" fashion, with the long axis of the molecule perpendicular to the heme plane. Remarkably, the structure and affinity of the complex are independent of ionic strength, indicating that there is little electrostatic interaction. Lowering the pH results in limited reorganization of the complex interface, while the binding affinity is unaffected. Therefore, protonation of the exposed copper ligand, His92, plays only a minor role in the complex. In contrast to other electron-transfer complexes, the plastocyanin-cytochrome f complex from P. laminosum is predominantly controlled by hydrophobic interactions. These findings are discussed in the context of the previously characterized angiosperm complex.

The role of charged residues on the surface of plastocyanin from the cyanobacterium Phormidium laminosum in the reaction with soluble cytochrome f in vitro was studied using site-directed mutagenesis. The charge on each of five residues on the eastern face of plastocyanin was neutralized and/or inverted, and the effect of the mutation on midpoint potentials was determined. The dependence of the overall rate constant of reaction, k(2), on ionic strength was investigated using stopped-flow spectrophotometry. Removing negative charges (D44A or D45A) accelerated the reaction and increased the dependence on ionic strength, whereas removing positive charges slowed it down. Two mutations (K46A, K53A) each almost completely abolished any influence of ionic strength on k(2), and three mutations (R93A, R93Q, R93E) each converted electrostatic attraction into repulsion. At low ionic strength, wild type and all mutants showed an inhibition which might be due to changes in the interaction radius as a consequence of ionic strength dependence of the Debye length or to effects on the rate constant of electron transfer, k(et). The study shows that the electrostatics of the interaction between plastocyanin and cytochrome f of P. laminosum in vitro are not optimized for k(2). Whereas electrostatics are the major contributor to k(2) in plants [Kannt, A., et al. (1996) Biochim. Biophys. Acta 1277, 115-126], this role is taken by nonpolar interactions in the cyanobacterium, leading to a remarkably high rate at infinite ionic strength (3.2 x 10(7) M(-1) s(-1)).

Cytochrome c6A is a unique dithio-cytochrome of green algae and plants. It has a very similar core structure to that of bacterial and algal cytochromes c6 but is unable to fulfill the same function of transferring electrons from cytochrome f to photosystem I. A key feature is that its heme midpoint potential is more than 200 mV below that of cytochrome c6 despite having His and Met as axial heme-iron ligands. To identify the molecular origins of the difference in potential, the structure of cytochrome c6 from the cyanobacterium Phormidium laminosum has been determined by X-ray crystallography and compared with the known structure of cytochrome c6A. One salient difference of the heme pockets is that a highly conserved Gln (Q51) in cytochrome c6 is replaced by Val (V52) in c6A. Using protein film voltammetry, we found that swapping these residues raised the c6A potential by +109 mV and decreased that of c6 by almost the same extent, -100 mV. X-ray crystallography of the V52Q protein showed that the Gln residue adopts the same configuration relative to the heme as in cytochrome c6 and we propose that this stereochemistry destabilizes the oxidized form of the heme. Consequently, replacement of Gln by Val was probably a key step in the evolution of cytochrome c6A from cytochrome c6, inhibiting reduction by the cytochrome b6f complex and facilitating establishment of a new function.