Abstract
A number of surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in amino acids located in the amino-terminal hydrophobic patch of the copper protein, which presents a variant structure as compared with other plastocyanins. The single mutants Y12G, Y12F, Y12W, P14L, and double mutant Y12G/P14L have been produced. Their reactivity toward photosystem I has been analyzed by laser flash absorption spectroscopy. Plots of the observed rate constant with all mutants versus plastocyanin concentration show a saturation profile similar to that with wild-type plastocyanin, thus suggesting the formation of a plastocyanin-photosystem I transient complex. The mutations do not induce relevant changes in the equilibrium constant for complex formation but induce significant variations in the electron transfer rate constant, mainly with the two mutants at proline 14. Additionally, molecular dynamics calculations indicate that mutations at position 14 yield small changes in the geometry of the copper center. The comparative kinetic analysis of the reactivity of plastocyanin mutants toward photosystem I from different organisms (plants and cyanobacteria) reveals that reversion of the unique proline of Prochlorothrix plastocyanin to the conserved leucine of all other plastocyanins at this position enhances the reactivity of the Prochlorothrix protein.
Highlights
Plastocyanin (Pc)1 is a small redox protein that functions in photosynthesis as a mobile electron carrier between the two membrane-embedded complexes cytochrome b6f and photosystem I (PSI) [1,2,3]
Changes have been made in amino acids located in the amino-terminal hydrophobic patch of the copper protein, which presents a variant structure as compared with other plastocyanins
Prochlorophytes represent a deeply branched group of cyanobacteria containing both chlorophyll a and b [10, 11]. These studies have shown that Prochlorothrix Pc reacts with PSI according to a two-step reaction mechanism involving complex formation and electron transfer, the complex being mainly hydrophobic in nature
Summary
Plastocyanin (Pc) is a small redox protein (molecular mass, ϳ10.5 kDa) that functions in photosynthesis as a mobile electron carrier between the two membrane-embedded complexes cytochrome b6f and photosystem I (PSI) [1,2,3]. Whereas higher plants produce just Pc, there is a number of intermediate species, both cyanobacteria and eukaryotic algae, that are able to synthesize cytochrome c6 as an alternative redox carrier under copper deficiency [4] The interaction between these two metalloproteins and PSI has been studied by laser-flash absorption spectroscopy in a wide variety of evolutionarily differentiated organisms, including prokaryotic and eukaryotic systems [5, 6]. Prochlorophytes represent a deeply branched group of cyanobacteria containing both chlorophyll a and b [10, 11] These studies have shown that Prochlorothrix Pc reacts with PSI according to a two-step reaction mechanism involving complex formation and electron transfer, the complex being mainly hydrophobic in nature. The laser-flash absorption spectroscopy analyses reported indicate that the replacement of Pro-14 by leucine, which is the “standard” residue in all other Pcs, makes the copper protein react much more efficiently with PSI
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