ABSTRACT
Oxidation of the soluble, truncated form of cytochrome f by wild-type and mutant species of plastocyanin has been analyzed by laser flash absorption spectroscopy in the cyanobacterium Nostoc (formerly, Anabaena) sp. PCC 7119. At low ionic strengths, the apparent electron transfer rate constant of cytochrome f oxidation by wild-type plastocyanin is 1.34 x 10(4) s(-)(1), a value much larger than those determined for the same proteins from other organisms. Upon site-directed mutagenesis of specific residues at the plastocyanin interaction area, the rate constant decreases in all cases yet to varying extents. The only exception is the D54K variant, which exhibits a higher reactivity toward cytochrome f. In most cases, the reaction rate constant decreases monotonically with an increase in ionic strength. The observed changes in the reaction mechanism and rate constants are in agreement with the location of the mutated residues at the interface area, as well as with the peculiar orientation of the two partners within the Nostoc plastocyanin-cytochrome f transient complex, whose NMR structure has been determined recently. Furthermore, the experimental data herein reported match well the kinetic behavior exhibited by the same set of plastocyanin mutants when acting as donors of electrons to photosystem I [Molina-Heredia, F. P., et al. (2001) J. Biol. Chem. 276, 601-605], thus indicating that the copper protein uses the same surface areas-one hydrophobic and the other electrostatic-to interact with both cytochrome f and photosystem I.
Subject(s)
Cytochromes f/metabolism , Lasers , Nostoc/metabolism , Plastocyanin/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Cytochromes f/chemistry , Cytochromes f/radiation effects , DNA Primers , Kinetics , Magnetic Resonance Spectroscopy , Models, Molecular , Mutagenesis, Site-Directed , Osmolar Concentration , Oxidation-Reduction , Plastocyanin/genetics , Protein Conformation , Recombinant Proteins/metabolismABSTRACT
Photosystem I reduction by the soluble metalloproteins cytochrome c(6) and plastocyanin, which are alternatively synthesized by some photosynthetic organisms depending on the relative availability of copper and iron, has been investigated in cyanobacteria, green algae and plants. The reaction mechanism is classified in three different types on the basis of the affinity of the membrane complex towards its electron donor protein. The role of electrostatic interactions in forming an intermediate transient complex, as well as the structural and functional similarities of cytochrome c(6) and plastocyanin are analysed from an evolutionary point of view. The proposal made is that the heme protein was first "discovered" by nature, when iron was much more abundant on the Earth's surface, and replaced by plastocyanin when copper became available because of the oxidizing conditions of the new atmosphere.
