Transient complex formation between plastocyanin from Prochlorothrix hollandica and cytochrome f from Phormidium laminosum was investigated using nuclear magnetic resonance (NMR) spectroscopy. Binding curves derived from NMR titrations at 10 mM ionic strength reveal a 1:1 stoichiometry and a binding constant of 6 (+/-2) x 10(3) M(-1) for complex formation, 1 order of magnitude larger than that for the physiological plastocyanin-cytochrome f complex from Ph. laminosum. Chemical-shift perturbation mapping indicates that the hydrophobic patch of plastocyanin is involved in the complex interface. When the unusual hydrophobic patch residues of P. hollandica plastocyanin were reverted to the conserved residues found in most other plastocyanins (Y12G/P14L), the binding constant for the interaction with cytochrome f was unaffected. However, the chemical shift perturbation map was considerably different, and the size of the average perturbation decreased by 40%. The complexes of both the wild-type and double mutant plastocyanin with cytochrome f were sensitive to ionic strength, contrary to the physiological complex. The possible implications of these findings for the mechanism of transient complex formation are discussed.

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. Plastocyanin (Pc)1 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 b 6 f and photosystem I (PSI) complexes (1, 2). In eukaryotic Pc, two active sites have been identified: site 1, an hydrophobic flat region around the copper binding area, and site 2, a charged region referred to as the acidic patch in plants and eukaryotic algae because it includes aspartate and glutamate residues. Whereas site 1 is involved in vitro in hydrophobic interactions of Pc with both redox partners and in the electron transfer event itself, site 2 is responsible for the electrostatic interactions and molecular recognition with complementary positive areas both in PSI and cytochrome f (3). However, the relevance of such electrostatic interactions in vivo can be different, as is the case for the interaction between cytochrome f and Pc (4). In cyanobacteria, site 2 can be either negatively or positively charged (5). All these differences between distinct organisms have led to different reaction mechanisms for PSI reduction (5-7).Prochlorophytes represent a diverse group of cyanobacteria containing both chlorophyll a and b (8, 9). Recently, an analysis of the interaction of Pc with PSI from the prochlorophyte Prochlorothrix hollandica, both for WT and mutated Pc (10, 11), revealed that Prochlorothrix Pc reacts with PSI by forming a transient complex with PSI that is stabilized by means of hydrophobic interactions. The...