Laser flash-induced kinetic analysis of cytochrome f oxidation by wild-type and mutant plastocyanin from the cyanobacterium Nostoc sp. PCC 7119.
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.
Albarrán
Cristina
C
Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, Universidad de Sevilla y Consejo Superior de Investigaciones Científicas, Sevilla, Spain.
Navarro
José A
JA
Molina-Heredia
Fernando P
FP
Murdoch
Piedad del S
Pdel S
De la Rosa
Miguel A
MA
Hervás
Manuel
M
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D045348:Cytochromes f / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D017931:DNA Primers; D007700:Kinetics; D007834:Lasers; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D046937:Nostoc / Q000378:metabolism*; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010970:Plastocyanin / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D010970:Plastocyanin; D045348:Cytochromes f