Abstract
Three surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in methionine 33, located in the hydrophobic patch of the copper protein, and in arginine 86 and proline 53, both located in the eastern hydrophilic area. The reactivity toward photosystem I of single mutants M33N, P53A, P53E, R86Q, R86E, and the double mutant M33N/P14L has been studied by laser flash absorption spectroscopy. All the mutations yield increased reactivity of plastocyanin toward photosystem I as compared with wild type plastocyanin, thus indicating that in Prochlorothrix electron donation to photosystem I is not optimized. The most drastic increases in the intracomplex electron transfer rate are obtained with mutants in methionine 33, whereas replacing arginine 86 only modestly affects the plastocyanin-photosystem I equilibrium constant for complex formation. Mutations at position 53 also promote major changes in the association of plastocyanin with photosystem I, yielding a change from a mechanism involving complex formation to a simpler collisional interaction. Molecular dynamics calculations indicate that mutations at position 33 promote changes in the H-bond network around the copper center. The comparative kinetic analysis of the reactivity of Prochlorothrix plastocyanin mutants toward photosystem I from other cyanobacteria reveals that mutations M33N, P53A, and P53E result in enhanced general reactivity.
Highlights
Plastocyanin (Pc)1 is a soluble type-I copper metalloprotein located inside the thylakoid lumen of photosynthetic organisms and acting as a mobile electron carrier between the membrane-embedded cytochrome b6f and photosystem I (PSI) complexes [1, 2]
The variant residue Met-33 at the hydrophobic patch of Prochlorothrix Pc (Fig. 1) was chosen to be mutated to the standard asparagine in other Pcs in order to study its role in the reactivity of Pc toward PSI, because it has been previously reported that replacement in this area of the atypical Pro-14 by the standard leucine drastically enhances Prochlorothrix Pc electron transfer to PSI [11]
The dependence of the observed pseudo first-order rate constant upon donor protein concentration shows a saturation profile for all mutants at positions 33 and 86 but not for those at position 53, which present linear concentration dependences (Fig. 3). These findings suggest the formation of a bimolecular (Pc-PSI) transient complex prior to electron transfer for mutants at positions 33 and 86, as previously described for the WT system as well as Midpoint redox potentials (Em ) of wild-type and mutant plastocyanins and association rate constants (KA ) and electron transfer rate constants for Prochlorothrix photosystem I reduction by the different mutants
Summary
Plastocyanin (Pc) is a soluble type-I copper metalloprotein (molecular mass, ϳ10.5 kDa) located inside the thylakoid lumen of photosynthetic organisms and acting as a mobile electron carrier between the membrane-embedded cytochrome b6f and photosystem I (PSI) complexes [1, 2]. A computational simulation of Prochlorothrix Pc-PSI docking suggested that Tyr-12 in Pc participates in hydrogen bonding with an asparagine residue in the PsaB polypeptide of PSI This model shows a short ␣-helix in Pc around position Pro-53 interacting with a small -sheet extension of the PsaA polypeptide in PSI [13]. We have extended our previous studies of Prochlorothrix PSI reduction by Pc to analyze the reactivity of Prochlorothrix Pc mutants at Met-33, in the hydrophobic patch, as well as at Pro-53 and Arg-86, in the electrostatically charged area. Mutagenesis of Prochlorothrix Plastocyanin replacing Met-33 by the standard asparagine (as is the case for the replacement of Pro-14 by leucine) as well as Pro-53 substitution make the copper protein react much more efficiently with PSI, whereas Arg-86 replacement only promotes moderate effects on (Pc-PSI) complex formation
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