Role of Electrostatics in the Interaction between Cytochromefand Plastocyanin of the CyanobacteriumPhormidium laminosum

Abstract

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)).

Transient Homodimer Interactions Studied Using the Electron Self-exchange Reaction

“…Similar conclusions have been made about the in vitro reactivity of PCu and cytochrome f from different organisms at physiological ionic strength (57) and electrostatics seem to be less important for this interaction in vivo (58). Taken together all these facts suggest the possibility of ESE facilitating intercomplex ET during photosynthesis.…”

Structure of the Complex between Plastocyanin and Cytochrome f from the Cyanobacterium Nostoc sp. PCC 7119 as Determined by Paramagnetic NMR

“…The closest charged residue of Cf (Glu-165) is 9 Å away. Thus, the electrostatic interaction between them may be moderate, and the kinetic effect of replacing Arg-93 could be attributable to more general electrostatic interactions, specifically during the encounter phase of the complex formation process, as described for the Arg-93 of P. laminosum Pc (28,29). The mutation L14A has been shown to have a large effect on the reactivity of Pc toward PSI (63).…”

“…The structure of the Phormidium complex does not exhibit a strong ionic strength dependence (31), and also the electron transfer kinetics show only a weak salt dependence (28,29). Both the plant and the Nostoc complexes employ electrostatics but with reverse charges.…”

“…Notably, the relevance of the electrostatic interactions could not be confirmed in vivo (15). In the system from Phormidium laminosum, the only cyanobacterium for which the kinetics of the reduction reaction have been analyzed so far, the electrostatic effects appear to be weaker and less optimized compared with plants (28,29).…”

Structure of the Intermolecular Complex between Plastocyanin and Cytochrome f from Spinach

“…As a result, one of the functionally relevant plant plastocyanins patches, the so-called acidic or eastern patch, is barely observed in the case of the bacterial proteins. These differences in surface charge properties have a profound influence on the kinetics of the interaction between Cyt f and Pc (29,30).…”