Abstract
Positively charged plastocyanin from Anabaena sp. PCC 7119 was investigated by site-directed mutagenesis. The reactivity of its mutants toward photosystem I was analyzed by laser flash spectroscopy. Replacement of arginine at position 88, which is adjacent to the copper ligand His-87, by glutamine and, in particular, by glutamate makes plastocyanin reduce its availability for transferring electrons to photosystem I. Such a residue in the copper protein thus appears to be isofunctional with Arg-64 (which is close to the heme group) in cytochrome c(6) from Anabaena (Molina-Heredia, F. P., Diaz-Quintana, A., Hervás, M., Navarro, J. A., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 33565-33570) and Synechocystis (De la Cerda, B., Diaz-Quintana, A., Navarro, J. A. , Hervás, M., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 13292-13297). Other mutations concern specific residues of plastocyanin either at its positively charged east face (D49K, H57A, H57E, K58A, K58E, Y83A, and Y83F) or at its north hydrophobic pole (L12A, K33A, and K33E). Mutations altering the surface electrostatic potential distribution allow the copper protein to modulate its kinetic efficiency: the more positively charged the interaction site, the higher the rate constant. Whereas replacement of Tyr-83 by either alanine or phenylalanine has no effect on the kinetics of photosystem I reduction, Leu-12 and Lys-33 are essential for the reactivity of plastocyanin.
Highlights
Their respective three-dimensional structures have repeatedly been solved using proteins from different organisms, and their structure-function relationships have been comparatively analyzed
We have recently reported that Cyt from Synechocystis [6] and Anabaena [7] possesses two areas equivalent to those of Pc: site 1, which is a hydrophobic region at the edge of the heme pocket providing the contact surface for electron transfer, and site 2, which is a charged patch driving the electrostatic movement toward its two membrane-anchored partners
Our mutagenesis studies of these two cyanobacterial Cyts revealed the existence of a highly conserved arginine residue at position 64, which is located on the protein surface at the frontier between sites 1 and 2, very close to the heme group, that is essential for electron transfer to photosystem I (PSI) [6, 7]
Summary
Their respective three-dimensional structures have repeatedly been solved using proteins from different organisms, and their structure-function relationships have been comparatively analyzed. The steric hindrance and nature of interactions at site 1 were investigated by replacing Leu-12 by Ala, and Lys-33 by Ala or Glu. The functional equivalence of site 2 in Anabaena Pc and in the negatively charged copper proteins was analyzed by substituting residues at positions 49, 57, and 58; Asp-49 was replaced by Lys, and both His-57 and Lys-58 were replaced by either Ala or Glu. Tyr-83 was changed to Ala and Phe to check whether Tyr-83 is involved in redox reactions, a role that was first proposed by He et al [24] but later discarded by Bendall et al [25] in other Pcs. Our analysis of all primary sequences of Pcs deposited at the Protein Data Bank with the BLAST search program [26] revealed that Arg-88 is conserved in cyanobacteria but not in higher plants or in green algae.
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call