Photosynthesis Research 57: 93–100, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands. 93 Regular paper The reaction mechanism of Photosystem I reduction by plastocyanin and cytochrome c 6 follows two different kinetic models in the cyanobacterium Pseudanabaena sp. PCC 6903 Manuel Herv´ as, Jos´ e A. Navarro, Fernando P. Molina-Heredia & Miguel A. De la Rosa ∗ Instituto de Bioqu´ ımica Vegetal y Fotos´ ıntesis, Centro ‘Isla de la Cartuja’, Universidad de Sevilla y CSIC, Am´ erico Vespucio s/n, 41092-Sevilla, Spain; ∗ Author for correspondence Received 28 January 1998; accepted in revised form 4 May 1998 Key words: cytochrome c 6 , electron transfer, Photosystem I, plastocyanin, Pseudanabaena Abstract Plastocyanin (Pc) and cytochrome c 6 (Cyt) have been purified to homogeneity from the cyanobacterium Pseudan- abaena sp. PCC 6903, which occupies a unique divergent branch in the evolutionary tree of oxygen-evolving photosynthetic organisms. The two metalloproteins have similar molecular masses (9–10 kDa), as well as almost identical isoelectric points (ca. 8) and midpoint redox potentials (ca. 350 mV, at pH 7). Their reaction mechanism of electron transfer to Photosystem I (PS I) has been analyzed by laser-flash absorption spectroscopy. The kinetic traces with Pc correspond to monophasic kinetics, whereas those with Cyt are better fitted to biphasic curves. The observed pseudo first-order rate constant (k obs ) with Pc and that for the slower phase with Cyt exhibit saturation profiles at increasing donor protein concentrations, thereby suggesting that the two metalloproteins are able to form transient complexes with PS I. The ionic strength dependence of the rate constants for complex formation makes evident the electrostatic nature of intermediate complexes. The experimental findings indicate that the PS I reduction kinetics in Pseudanabaena follow a type II mechanism with Pc and a type III mechanism with Cyt, according to the different kinetic models proposed previously [(Hervás M, Navarro JA, Díaz A, Bottin H and De la Rosa MA (1995) Biochemistry 34: 11321–11326)]. From an evolutionary point of view, this reinforces our previous observation that PS I was first adapted to operate efficiently with positively charged Cyt rather than with Pc. Abbreviations: Cyt – cytochrome c 6 ;E m – midpoint redox potential; K A – equilibrium constant for the complex formation reaction; k et – electron transfer first-order rate constant; k obs – observed pseudo first-order rate constant; K R – equilibrium constant for rearrangement of redox proteins within the reaction complex; Pc – plastocyanin; pI, isoelectric point; PS I – Photosystem I; G et , H et , S et – activation free energy, enthalpy and entropy of electron transfer Introduction Plastocyanin (Pc) and cytochrome c 6 (Cyt) are two small redox proteins (molecular masses, ca. 10.5 and 9.0 kDa, respectively) that function as mobile electron carriers between two membrane-embedded complexes: cytochrome b 6 f and Photosystem I (PS I) (see Chitnis et al. 1995; Navarro et al. 1997, for reviews). Whereas some cyanobacteria only syn- thesize Cyt and higher plants produce just Pc, there is a number of intermediate species – both cyanobacteria and eukaryotic algae – that are able to form either Cyt or Pc (Ho and Krogmann 1984). The two proteins are acidic in eukaryotic organ- isms and either neutral or basic in cyanobacteria, but they have an almost identical isoelectric point when isolated from the same source (Hervás et al. 1995).