Laser Flash-Induced Kinetic Analysis of Cytochrome f Oxidation by Wild-Type and Mutant Plastocyanin from the Cyanobacterium Nostoc sp. PCC 7119 ² Cristina Albarra ´n, Jose ´ A. Navarro, Fernando P. Molina-Heredia, Piedad del S. Murdoch, Miguel A. De la Rosa, and Manuel Herva ´s* Instituto de Bioquı ´mica Vegetal y Fotosı ´ntesis, Centro de InVestigaciones Cientı ´ficas Isla de la Cartuja, UniVersidad de SeVilla y Consejo Superior de InVestigaciones Cientı ´ficas, SeVilla, Spain ReceiVed May 17, 2005; ReVised Manuscript ReceiVed June 29, 2005 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 × 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 areassone hydrophobic and the other electrostaticsto interact with both cytochrome f and photosystem I. Cytochrome (Cyt) 1 f (33 kDa), a c-type Cyt, is one of the four integral redox components of the Cyt b 6 f complex of the thylakoidal membrane in oxygenic photosynthetic organ- isms, where it accepts electrons from the Rieske iron-sulfur protein and passes them to P 700 + in photosystem I (PSI) via the luminal electron carriers plastocyanin (Pc) and Cyt c 6 (1, 2). Cyt f has been purified from a variety of plant, algal, and cyanobacterial sources, and its overall three-dimensional structure has shown to be highly conserved (3-7). However, the surface electrostatic potential of Cyt f varies considerably from one organism to another, the protein being highly positive in eukaryotes but slightly negative in cyanobacteria (1, 3). In fact, a conserved basic ridge is present in plant and algal Cyt f, but is replaced with more diffusely arranged acidic residues in cyanobacterial proteins. Because of the low water solubility of native Cyt f, the soluble truncated forms of the protein from different sources have been used with great success to perform both structural and functional in vitro studies (6-13). Extensive kinetic and structural analyses of the transient Pc-Cyt f complex have been carried out in an ample variety of organisms, with relevant differences being observed with regard to the reaction mechanism and relative orientation of the two partners inside the complex (8, 9). First, the redox interaction is mainly hydrophobic in the cyanobacterium Phormidium laminosum (8, 9) but electrostatically driven in plants. Second, Pc binds to Cyt f in a “head-on” conformation in Phormidium, in which the hydrophobic patch of Pc (the so-called site 1) accounts for the whole recognition interface area, but in a “side-on” orientation in plants, with the acidic patch of Pc (so-called site 2) being involved as well (see ref 9 for a recent review). Pc and Cyt f from the cyanobacterium Nostoc sp. PCC 7119, in particular, form a highly interesting system as they react with each other by means of attractive electrostatic interactions, like plant proteins, but using differently charged patches, which are positive in Pc and negative in Cyt f. Noteworthy is the fact that the three-dimensional structure of the Nostoc Pc-Cyt f complex has been recently character- ized by NMR spectroscopy (14), thus revealing significant differences regarding the relative orientation of both proteins within the complex as compared with that previously reported for plants and Phormidium. Actually, the conformation of the Nostoc Pc-Cyt f complex resembles the characteristic ² This work was supported by grants from the European Commission (HPRN-CT1999-00095), the Spanish Ministry of Education, Culture and Sport (AP2001-1256), the Spanish Ministry of Science and Technology (BMC2003-00458), and the Andalusian Government (PAI, CVI-0198). * To whom correspondence should be addressed: Instituto de Bioquı ´mica Vegetal y Fotosı ´ntesis, Universidad de Sevilla y CSIC, Ame ´rico Vespucio 49, 41092 Sevilla, Spain. Telephone: 34-954-489- 514. Fax: 34-954-460-065. E-mail: mhervas@us.es. 1 Abbreviations: Cyt, cytochrome; dRf, deazariboflavin; k2, second- order rate constant; KA, equilibrium constant for complex formation; k′et, apparent electron transfer rate constant; kobs, observed pseudo-first- order rate constant; Pc, plastocyanin; PDQ, propylendiquat; PSI, photosystem I; WT, wild-type. 11601 Biochemistry 2005, 44, 11601-11607 10.1021/bi050917g CCC: $30.25 © 2005 American Chemical Society Published on Web 08/02/2005