Fast Electron Transfer from Cytochrome c 6 and Plastocyanin to Photosystem I of Chlamydomonas reinhardtii Requires PsaF † Michael Hippler, ‡ Friedel Drepper, § Joseph Farah, ‡ and Jean-David Rochaix* ,‡ Departments of Molecular Biology and Plant Biology, UniVersity of GeneVa, 30 Quai Ernest Ansermet, CH-1211 GeneVa, Switzerland, and De ´ partement de Biologie Cellulaire et Mole ´ culaire, CEA-Saclay, 91191 Gif sur YVette Cedex, France ReceiVed January 14, 1997; ReVised Manuscript ReceiVed March 25, 1997 X ABSTRACT: To study the function of the PsaF subunit of photosystem I (PSI), the interactions between plastocyanin or cytochrome c 6 and PSI isolated from wild-type and a PsaF-deficient mutant of Chlamydomonas reinhardtii have been examined using cross-linking techniques and flash absorption spectroscopy. We show that efficient electron transfer from both plastocyanin and cytochrome c 6 to PSI depends on PsaF. A remarkable feature of the PSI complex of C. reinhardtii is that both plastocyanin and cytochrome c 6 reduce P700 + with first-order kinetics and a half-time of 3 µs, which is unique among photosynthetic organisms examined. The photosystem I (PSI) 1 complex functions as a light- driven oxidoreductase that transfers electrons from plasto- cyanin to ferredoxin in higher plants, most algae, and cyanobacteria. In some cyanobacteria and algae, the type I copper protein plastocyanin can be replaced by a class I c-type cytochrome, depending on the relative availability of copper and iron in the culture medium (Wood, 1978; Ho & Krogmann, 1984; Sandmann, 1986; Merchant & Bogorad, 1986). The PSI reaction center is a membrane-bound complex consisting of 13-14 polypeptide subunits. The three-dimensional structure of PSI from the cyanobacterium Synechococcus elongatus has been determined by X-ray crystallography at a resolution of 6 Å (Krauss et al., 1993) and more recently of 4 Å (Krauss et al., 1996). P700 of PSI is localized near the lumenal surface of the thylakoid membrane and is therefore accessible to the lumenal electron donor proteins plastocyanin and cytochrome c 6 . Cross- linking results suggest that PsaF is involved in docking of plastocyanin and cytochrome c 6 to the PSI complex (Wynn & Malkin, 1988; Wynn et al., 1989; Hippler et al., 1989). The conformations of the cross-linked and authentic plas- tocyanin-PSI complexes appear to be similar based on the fast kinetics of reduction of P700 + with a half-time of 13- 15 µs observed in the cross-linked complex (Hippler et al., 1989). This half-time is comparable to that found in intact thylakoids (Haehnel & Witt, 1971; Haehnel et al., 1989), with digitonin-PSI particles (Bottin & Mathis, 1985, 1987), and with PSI-200 particles (Drepper et al., 1996) at high plastocyanin concentrations. The function of the PsaF subunit in PSI remains elusive [see Golbeck (1992)]. This subunit was found to be associated with the LHCI complex of PSI (Anandan et al., 1989; Bassi et al., 1992; Scheller & Møller, 1990). Removal of the PsaF subunit from a plant-derived PSI complex by mild detergent treatment impairs the electron transfer from plastocyanin to PSI (Bengis & Nelson, 1977). Surprisingly, the specific deletion of the psaF gene in cyanobacteria did not affect photoautotrophic growth (Chitnis et al., 1991), and the in ViVo measured electron transfer rate between cyto- chrome c 553 and PSI was the same as in wild-type (Xu et al., 1994). In contrast, the electron transfer reaction between plastocyanin and P700 + was shown to be considerably reduced within whole cells of the 3bF strain of Chlamy- domonas reinhardtii which lacks the psaF gene (Farah et al., 1995). Mass spectroscopic analysis of tryptic peptides of plastocyanin and of the cross-linked product of plasto- cyanin and PsaF from spinach revealed that the PsaF subunit appears to be cross-linked with one of its N-terminal Lys residues to the conserved acidic amino acids 42-44 and 59- 61 of plastocyanin (Hippler et al., 1996). A region close to the N-terminal end of PsaF could form an amphipathic R-helix, whose positively charged face may interact with plastocyanin (Hippler et al., 1996). The half-time of fast electron transfer between plastocyanin and PSI in the unicellular alga Chlorella was measured to be 4 µs (Delosme, 1991), whereas no corresponding fast phase could be observed in the cyanobacterium Synechocystis (Herva `s et al., 1994) and in the thermophilic cyanobacterium Synechococcus elongatus (Hatanaka et al., 1993). Since the PsaF subunits of these cyanobacteria lack the positively charged N-proximal region [for amino acid sequences, see Hippler et al. (1996)], the question arises whether this segment evolved to allow fast electron transfer between plastocyanin and PSI in higher plants and algae. The electron transfer from cytochrome c 6 to P700 + was found to display a first-order kinetic component with a half- time of 8 µs in the green alga Monoraphidium braunii and of 4 µs in the cyanobacterium Anabaena sp. PCC7119, whereas no first-order microsecond phase could be detected † This work was supported by a grant from the Human Frontier Science Program and by Grant 31.34014.92 from the Swiss National Fond. F.D. and M.H. were supported by a long-term fellowship from the European Community and from the Human Frontier Science Program, respectively. * Address correspondence to this author. ‡ University of Geneva. § CEA-Saclay. X Abstract published in AdVance ACS Abstracts, May 1, 1997. 1 Abbreviations: chl, chlorophyll; cytc, cytochrome c6; FWHM, full width at half-maximum; Mops, 3-(N-morpholino)propanesulfonic acid; pc, plastocyanin; PSI, photosystem I. 6343 Biochemistry 1997, 36, 6343-6349 S0006-2960(97)00082-2 CCC: $14.00 © 1997 American Chemical Society