The Specificity in the Interaction between Cytochrome f and Plastocyanin from the Cyanobacterium Nostoc sp. PCC 7119 Is Mainly Determined by the Copper Protein

Abstract

The plastocyanin-cytochrome f complex from Nostoc exhibits relevant structural differences when compared with the homologous complexes from other cyanobacteria and plants, with electrostatic and hydrophobic interactions being differently involved in each case. Here, five negatively charged residues of a recombinant form of cytochrome f from Nostoc have been replaced with either neutral or positively charged residues, and the effects of mutations on the kinetics of electron transfer to wild-type and mutant forms of plastocyanin have been measured by laser flash absorption spectroscopy. Cytochrome f mutants with some negative charges replaced with neutral residues exhibit an apparent electron transfer rate constant with wild-type plastocyanin similar to or slightly higher than that of the wild-type species, whereas the mutants with negative charges replaced with positive residues exhibit a significantly lower reactivity. Taken together, these results indicate that the effects of neutralizing residues at the electrostatically charged patch of cytochrome f are smaller than those previously observed for mutants of plastocyanin, thus suggesting that it is the copper protein which determines the specificity of the electrostatic interaction with the heme protein. Moreover, cross reactions between mutants of both proteins reveal the presence of some short-range specific electrostatic interactions. Our findings also make evident the fact that in Nostoc the main contribution to the electrostatic nature of the complex is provided by the small domain of cytochrome f.

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

“…21,22 Clearly, the hydrophobic patch plays a fundamental role in the formation of the Cyt f-Pc complexes. The CSP map is qualitatively similar to that of the N−N complex 22,27 with a prominent perturbation for R93, known to be involved in the binding in both the N−N complex 18,19,22 and Ph-Ph complex. 16 Interestingly, a significant CSP was also observed for K46 in the N-Ph complex.…”

The Complex of Cytochrome f and Plastocyanin from Nostoc sp. PCC 7119 is Highly Dynamic

“…The solution models of the complexes have been determined by rigid-body docking of the structures of the individual proteins on the basis of binding chemical shift perturbations and intermolecular pseudocontact shifts (PCSs) of Pc nuclei induced by the paramagnetic, oxidized iron of Cyt f [10][18] or negative charges on Cyt f [19] resulted in a decreased association rate constant. It could be shown that for Pc several charges are pivotal for the interaction [18 , 19].…”

“…In the case of the Nostoc complex, site-directed mutagenesis studies on the influence of charges on the kinetics of complex formation highlighted how the loss of either positive charges on Pc[18] or negative charges on Cyt f [19] resulted in a decreased association rate constant. It could be shown that for Pc several charges are pivotal for the interaction [18 , 19]. On the other hand, the charges on Cyt f are more spread out over the surface, and no “hot spots” were identified in either Nostoc [19] or P. laminosum, [15] thus suggesting that the encounter state might have an important role in these complexes.…”

Electrostatic strain and concerted motions in the transient complex between plastocyanin and cytochrome f from the cyanobacterium Phormidium laminosum

“…Pc is a small, soluble cupredoxin that shuttles electrons from Cf to photosystem I [2,3]. The structure of Pc is a betabarrel with two functional surface areas [2,3,[7][8][9]: one hydrophobic surrounding a copper-ligand histidine (site 1), and the other electrostatically charged (site 2). Cf, in its turn, is a subunit of cytochrome b 6 f [6] that is anchored to this membrane complex by its hydrophobic, C-terminal alpha-helix.…”

“…The electron transfer from Cf to Pc has been analysed by using stopped-flow [11][12][13][14][15] and fast kinetic techniques [7,16], often combined with site-directed mutagenesis of one or both partners. The resulting kinetic data have been very useful to analyse the interaction between Pc and Cf by means of theoretical methods, including Molecular Dynamics (MD) at sub-nanosecond time scale [17], rigid-body MD [18], Molecular Modelling [19] supported by NMR data and Brownian Dynamics (BD) simulations [20][21][22][23][24][25][26][27][28].…”