Hydrophobic Interactions in a Cyanobacterial Plastocyanin−Cytochrome f Complex

Abstract

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.

Different Interaction Modes of Two Cytochrome-c Oxidase Soluble CuA Fragments with Their Substrates

“…This is followed by a second step where the final (electron transfer) complex is generated mostly by specific, non-ionic contacts. A similar situation could be found for a cytochrome f-plastocyanin electron transfer couple from the cyanobacterium Phormidium laminosum, which has also been characterized by NMR (35) and double mutant cycle analysis by stopped-flow spectroscopy (36). Fig.…”

The Interactions of Cyanobacterial Cytochromec6 and Cytochrome f, Characterized by NMR

“…Titration curves of ⌬␦ Bind versus the molar ratio of cytf:Anacytc 6 were plotted for the 15 N nuclei of the four most strongly shifted resonances (Fig. 4A).…”

“…A similar expression system in E. coli JM109 was used for the production of uniformly 15 N-labeled Ana-cytc 6 . The culture was grown in M9 minimal media additionally supplemented with 1 g/liter 15 NH 4 Cl and 1 mM thiamine. Aerobic growth was maintained at 37°C for 72 h before harvesting.…”

“…Isolation and purification of Ana-cytc 6 were achieved as described previously (8,18). The preparation and purification of 15 N, 13 C double-labeled Syn-cytc 6 have been reported previously (21).…”

“…Protein Preparation-The soluble fragment of cytf was prepared according to previously published methods (15,16). Unlabeled Anacytc 6 was produced in Escherichia coli GM119 (17) transformed with both pEAC-WT (18) and pEC86 (19).…”