Nar1p, a conserved eukaryotic protein with similarity to Fe-only hydrogenases, functions in cytosolic iron-sulphur protein biogenesis.

The genome of the yeast Saccharomyces cerevisiae encodes the essential protein Nar1p that is conserved in virtually all eukaryotes and exhibits striking sequence similarity to bacterial iron-only hydrogenases. Previously, we have shown that Nar1p is an Fe-S protein and that assembly of its co-factors depends on the mitochondrial Fe-S cluster biosynthesis apparatus. Using functional studies in vivo, we demonstrated that Nar1p has an essential role in the maturation of cytosolic and nuclear, but not of mitochondrial, Fe-S proteins. Here we provide further spectroscopic evidence that Nar1p possesses two Fe-S clusters. We also show that Nar1p is required for Fe-S cluster assembly on the P-loop NTPase Nbp35p, another newly identified component of the cytosolic Fe-S protein assembly machinery. These data suggest a complex biochemical pathway of extra-mitochondrial Fe-S protein biogenesis involving unique eukaryotic proteins.

Daddy, where did (PS)I come from?

The reacton centre I (RCI)-type photosystems from plants, cyano-, helio- and green sulphur bacteria are compared and the essential properties of an archetypal RCI are deduced. Species containing RCI-type photosystems most probably cluster together on a common branch of the phylogenetic tree. The predicted branching order is green sulphur, helio- and cyanobacteria. Striking similarities between RCI- and RCII-type photosystems recently became apparent in the three-dimensional structures of photosystem I (PSI), PSII and RCII. The phylogenetic relationship between all presently known photosystems is analysed suggesting (a) RCI as the ancestral photosystem and (b) the descendence of PSII from RCI via gene duplication and gene splitting. An evolutionary model trying to rationalise available data is presented.

An essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic Fe/S proteins.

Biogenesis of Fe/S clusters involves a number of essential mitochondrial proteins. Here, we identify the essential Erv1p of Saccharomyces cerevisia mitochondria as a novel component that is specifically required for the maturation of Fe/S proteins in the cytosol, but not in mitochondria. Furthermore, Erv1p was found to be important for cellular iron homeostasis. The homologous mammalian protein ALR ('augmenter of liver regeneration'), also termed hepatopoietin, can functionally replace defects in Erv1p and thus represents the mammalian orthologue of yeast Erv1p. Previously, a fragment of ALR was reported to exhibit an activity as an extracellular hepatotrophic growth factor. Both Erv1p and full-length ALR are located in the mitochondrial intermembrane space and represent the first components of this compartment with a role in the biogenesis of cytosolic Fe/S proteins. It is likely that Erv1p/ALR operates downstream of the mitochondrial ABC transporter Atm1p/ABC7/Sta1, which also executes a specific task in this essential biochemical process.

Yeast ERV2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family.

Saccharomyces cerevisiae Erv2p was identified previously as a distant homologue of Erv1p, an essential mitochondrial protein exhibiting sulfhydryl oxidase activity. Expression of the ERV2 (essential for respiration and vegetative growth 2) gene from a high-copy plasmid cannot substitute for the lack of ERV1, suggesting that the two proteins perform nonredundant functions. Here, we show that the deletion of the ERV2 gene or the depletion of Erv2p by regulated gene expression is not associated with any detectable growth defects. Erv2p is located in the microsomal fraction, distinguishing it from the mitochondrial Erv1p. Despite their distinct subcellular localization, the two proteins exhibit functional similarities. Both form dimers in vivo and in vitro, contain a conserved YPCXXC motif in their carboxyl-terminal part, bind flavin adenine dinucleotide (FAD) as a cofactor, and catalyze the formation of disulfide bonds in protein substrates. The catalytic activity, the ability to form dimers, and the binding of FAD are associated with the carboxyl-terminal domain of the protein. Our findings identify Erv2p as the first microsomal member of the Erv1p/Alrp protein family of FAD-linked sulfhydryl oxidases. We propose that Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential, FAD-dependent oxidase of protein disulfide isomerase.

Biogenesis of iron-sulfur proteins in eukaryotes: a novel task of mitochondria that is inherited from bacteria.

Fe/S clusters are co-factors of numerous proteins with important functions in metabolism, electron transport and regulation of gene expression. Presumably, Fe/S proteins have occurred early in evolution and are present in cells of virtually all species. Biosynthesis of these proteins is a complex process involving numerous components. In mitochondria, this process is accomplished by the so-called ISC (iron-sulfur cluster assembly) machinery which is derived from the bacterial ancestor of the organelles and is conserved from lower to higher eukaryotes. The mitochondrial ISC machinery is responsible for biogenesis iron-sulfur proteins both within and outside the organelle. Maturation of the latter proteins involves the ABC transporter Atm1p which presumably exports iron-sulfur clusters from the organelle. This review summarizes recent developments in our understanding of the biogenesis of iron-sulfur proteins both within bacteria and eukaryotes.

Mitochondrial Isa2p plays a crucial role in the maturation of cellular iron-sulfur proteins.

The assembly of iron-sulfur (Fe/S) clusters in a living cell is mediated by a complex machinery which, in eukaryotes, is localised within mitochondria. Here, we report on a new component of this machinery, the protein Isa2p of the yeast Saccharomyces cerevisiae. The protein shares sequence similarity with yeast Isa1p and the bacterial IscA proteins which recently have been shown to perform a function in Fe/S cluster biosynthesis. Like the Isa1p homologue, Isa2p is localised in the mitochondrial matrix as a soluble protein. Deletion of the ISA2 gene results in the loss of mitochondrial DNA and a strong growth defect. Simultaneous deletion of the ISA1 gene does not further exacerbate this growth phenotype suggesting that the Isa proteins perform a non-essential function. When Isa2p was depleted by regulated gene expression, mtDNA was maintained, but cells grew slowly on non-fermentable carbon sources. The maturation of both mitochondrial and cytosolic Fe/S proteins was strongly impaired in the absence of Isa2p. Thus, Isa2p is a new member of the Fe/S cluster biosynthesis machinery of the mitochondrial matrix and may be involved in the binding of an intermediate of Fe/S cluster assembly.

The FAPY-DNA glycosylase (Fpg) is required for survival of the cyanobacterium Synechococcus elongatus under high light irradiance.

The gene for the DNA repair enzyme Fpg from Synechococcus elongatus was detected immediately downstream of the photosystem I gene psaE. fpg is likely expressed together with psaE by transcriptional readover while psaE is mostly expressed independently. Segregated psaE and fpg deletion strains were obtained upon insertional inactivation of both genes in S. elongatus. These mutants are viable under photoautotrophic conditions, but fail to grow under high light regimes that likely cause oxidative stress. These high light sensitive phenotypes suggest that the Fpg protein, which has been shown to repair DNA lesions caused by reactive oxygen species in Escherichia coli, may be involved in the photoprotection of cyanobacteria against oxidative damage caused under high irradiance.

Sequence of the two operons encoding the four core subunits of the cytochrome b(6)f complex from the thermophilic Cyanobacterium synechococcus elongatus.

The genes encoding cytochrome f (petA), cytochrome b(6) (petB), the Rieske FeS-protein (petC), and subunit IV (petD) of the cytochrome b(6)f complex from the thermophilic cyanobacterium Synechococcus elongatus were cloned and sequenced. Similar to other cyanobacteria, the structural genes are arranged in two short, single-copy operons, petC/petA and petB/petD, respectively. In addition, five open reading frames with homology to known orfs from the cyanobacterium Synechocystis PCC 6803 were identified in the immediate vicinity of these two operons.

Lys35 of PsaC is required for the efficient photoreduction of flavodoxin by photosystem I from Chlamydomonas reinhardtii.

The photoreduction of the oxidized and the semiquinone form of flavodoxin from Synechocystis sp. PCC 6803 by the photosystem I (PSI) of wild-type Chlamydomonas reinhardtii and the mutant strains Lys35Asp, Lys35Glu and Lys35Arg was analysed by flash-absorption spectroscopy to investigate the role of residue Lys35 of the PSI subunit PsaC in flavodoxin reduction. For PSI preparations from C. reinhardtii the reduction of oxidized flavodoxin was monoexponential and approached limiting electron transfer rates similar to those of cyanobacterial PSI from the wild-type and the Lys35Arg mutant. For PSI from the Lys35Glu mutant, however, a approximately 2.5-fold smaller value was determined. The photoreduction of flavodoxin semiquinone by PSI from C. reinhardtii lacked fast first-order kinetic components and, in contrast with PSI from cyanobacteria, displayed only a single concentration-dependent phase. From this phase, second-order rate constants were calculated for wild-type PSI and PSI from the Lys35Arg mutant which were comparable to those of PSI from cyanobacteria. For PSI from the Lys35Glu and the Lys35Asp mutants the derived second-order rate constants were 19 and 10 times smaller. Thus, the inversion of charge at position 35 of PsaC negatively affects the rate of electron transfer to both forms of flavodoxin, whereas PSI complexes that retain a positive charge at this position show wild-type kinetics. However, the positive charge at this position of PsaC is not essential for flavodoxin photoreduction as the number of flavodoxin molecules reduced per PSI was similar for all of the PSI complexes investigated. In addition, chemical cross-linking assays showed that the binary cross-linking product between flavodoxin and PsaC of PSI from wild-type C. reinhardtii was not formed with PSI complexes from the Lys13Asp and Lys35Glu mutants. This indicates that Lys35 of PsaC is probably essential for the chemical cross-link between PsaC and flavodoxin. Taken together, these experiments show that Lys35 of PsaC plays a strikingly similar role in the electron transfer from PSI to both ferredoxin and flavodoxin.

Insertion of the N-terminal part of PsaF from Chlamydomonas reinhardtii into photosystem I from Synechococcus elongatus enables efficient binding of algal plastocyanin and cytochrome c6.

A strain of the cyanobacterium Synechococcus elongatus was generated that expresses a hybrid version of the photosystem I subunit PsaF consisting of the first 83 amino acids of PsaF from the green alga Chlamydomonas reinhardtii fused to the C-terminal portion of PsaF from S. elongatus. The corresponding modified gene was introduced into the genome of the psaF-deletion strain FK2 by cointegration with an antibiotic resistance gene. The transformants express a new PsaF subunit similar in size to PsaF from C. reinhardtii that is assembled into photosystem I (PSI). Hybrid PSI complexes isolated from these strains show an increase by 2 or 3 orders of magnitude in the rate of P700(+) reduction by C. reinhardtii cytochrome c6 or plastocyanin in 30% of the complexes as compared with wild type cyanobacterial PSI. The corresponding optimum second-order rate constants (k2 = 4.0 and 1.7 x 10(7) M1 s1 for cytochrome c6 and plastocyanin) are similar to those of PSI from C. reinhardtii. The remaining complexes are reduced at a slow rate similar to that observed with wild type PSI from S. elongatus and the algal donors. At high concentrations of C. reinhardtii cytochrome c6, a fast first-order kinetic component (t(1)/(2) = 4 microseconds) is revealed, indicative of intramolecular electron transfer within a complex between the hybrid PSI and cytochrome c6. This first-order phase is characteristic for P700(+) reduction by cytochrome c6 or plastocyanin in algae and higher plants. However, a similar fast phase is not detected for plastocyanin. Cross-linking studies show that, in contrast to PSI from wild type S. elongatus, the chimeric PsaF of PSI from the transformed strain cross-links to cytochrome c6 or plastocyanin with a similar efficiency as PsaF from C. reinhardtii PSI. Our data indicate that development of a eukaryotic type of reaction mechanism for binding and electron transfer between PSI and its electron donors required structural changes in both PSI and cytochrome c6 or plastocyanin.

Identification of biotinylation sites on proteins by selective retrieval of 2-iminobiotinylated peptides from proteolytic peptide mixtures: localization of the accessible lysine residues on the photosystem I subunits PsaD and PsaE.

An affinity purification technique was established that allows the selective isolation of 2-iminobiotinylated peptides from proteolytic digest of proteins in order to identify surface-exposed protein domains. Serving as model systems, two photosystem I subunits, PsaD and PsaE from the cyanobacterium Synechococcus elongatus, were overexpressed in Escherichia coli, modified in vitrowith NHS-2-iminobiotin which incorporates 2-iminobiotin at exposed amino groups, and subjected to proteolytic digestion by Glu-C and Arg-C protease, respectively. 2-Iminobiotin-containing proteolytic peptides were subsequently extracted from the proteolytic digests using avidin agarose in a batch procedure and the extracted peptides were separated by HPLC chromatography. The analysis of the peptide maps by electrospray ionization mass spectrometry or N-terminal sequencing showed that avidin-extracted peptide fractions contain almost exclusively 2-iminobiotinylated proteolytic fragments of PsaE or PsaD. No unmodified peptides of PsaD or PsaE were detected. According to this analysis, PsaE is accessible to biotinylation at all of its 7 lysine residues and at its N-terminus. Similarly, all 11 lysine residues of PsaD can be biotinylated and only the N-terminus of PsaD is not accessible.

The PsaE subunit is required for complex formation between photosystem I and flavodoxin from the cyanobacterium Synechocystis sp. PCC 6803.

The photoreduction of the oxidized and the semiquinone form of flavodoxin by photosystem I particles (PSI) from the wild type and a psaE deletion strain from the cyanobacterium Synechocystis sp. PCC 6803 was analyzed by flash-absorption spectroscopy to investigate a possible involvement of the PsaE subunit in this photoreduction process. The kinetics of the reduction of oxidized flavodoxin display a single-exponential component for both PSI preparations. Limiting electron transfer rates kobs of approximately 500 and approximately 900 s -1 are deduced for the wild type and PSI from the psaE-less mutant, respectively, indicating that the PsaE subunit is not important for this photoreduction process. In the case of wild-type PSI, the reduction of flavodoxin semiquinone is a biphasic process, displaying a fast first-order phase with a t1/2 of approximately 13 micro(s) which is then followed by a slower, concentration-dependent phase, for which a second-order rate constant k2 of 2.2 x 10(8) M-1 cm-1 is calculated. In contrast, photoreduction of the semiquinone by PSI from the psaE-less mutant is monoexponential, displaying only one second-order component with a second-order rate constant similar to those observed for wild-type PSI (k2 = 1.5 x 10(8) M-1 cm-1). The fast first-order component which is interpreted as an electron transfer process within a preformed complex between flavodoxin semiquinone and PSI is almost completely absent in the reduction of flavodoxin by the PsaE-less PSI. A similar loss of the fast phase is also observed for the photoreduction of flavodoxin semiquinone by PSI from a Synechococcus elongatus psaE-less mutant. Upon reconstitution of isolated PsaE to the PsaE-less PSI in vitro, approximately 80% of the fast first-order kinetic component is recovered, indicating that PsaE is required for high-affinity binding of the flavodoxin semiquinone to PSI. In addition, chemical cross-linking assays show that flavodoxin can no longer be cross-linked to PSI in detectable amounts when PsaE is missing on the reaction center. Taken together, these experiments indicate that the PsaE subunit is required for complex formation between PSI and flavodoxin but is not required for an efficient forward electron transfer from photosystem I to both forms of flavodoxin.

Characterization of the unbound 2[Fe4S4]-ferredoxin-like photosystem I subunit PsaC from the Cyanobacterium synechococcus elongatus.

Recombinant PsaC was reconstituted in vitro and investigated by UV/vis, EPR, and 1H NMR spectroscopy. Its UV/vis and EPR spectroscopic properties correspond to those of the wild-type protein. Fast repetition 1D and 2D 1H NMR spectra allowed the sequence-specific assignment of the hyperfine-shifted proton resonances of the cluster-ligating resonances, taking advantage also of chemical shift analogies with other 4 and 8 Fe ferredoxins and a structural model for PsaC. The Calpha-Cbeta-S-Fe dihedral angles of the cluster ligands could be estimated from the chemical shifts and relaxation properties of their betaCH2 protons. All NMR-derived structural information on PsaC confirms its similarity to smaller 8Fe ferredoxins serving as electron transfer proteins in solution. Partial reduction of PsaC leads to an intermediate species with strongly exchange broadened 1H NMR resonances. The intermolecular electron exchange rate is estimated to be in the 10(2)-10(4) s-1 range, the intramolecular electron exchange rate between the two [Fe4S4] clusters to be higher than 10(4) s-1. The consequences of these findings for the electron transfer in photosystem I are discussed.

Gene transfer and manipulation in the thermophilic cyanobacterium Synechococcus elongatus.

DNA can be introduced into the thermophilic cyanobacterium Synechococcus elongatus by electroporation or conjugation. Its genome can be readily manipulated through integrative transformation or by using promiscuous RSF1010-derived plasmids that can be transferred unaltered between Escherichia coli and Synechococcus elongatus. These vectors can therefore be used for in vivo studies of cyanobacterial proteins in both mesophilic and thermophilic cyanobacterial backgrounds. As a preliminary step towards the analysis of structure-function relationships of photosystem I (PSI) from this thermophile, the genes encoding the PSI subunits PsaF, PsaL, and PsaK were inactivated and shown to be non-essential in S. elongatus. In addition, PSI reaction centres were extracted from a psaL- strain exclusively as monomeric complexes.

Characterization of a redox-active cross-linked complex between cyanobacterial photosystem I and its physiological acceptor flavodoxin.

A covalent complex between photosystem I and flavodoxin from the cyanobacterium Synechococcus sp. PCC 7002 was generated by chemical cross-linking. Laser flash-absorption spectroscopy indicates that the bound flavodoxin of this complex is stabilized in the semiquinone state and is photoreduced to the quinol form upon light excitation. The kinetics of this photoreduction process, which takes place in approximately 50% of the reaction centres, displays three exponential components with half-lives of 9 microsec, 70 microsec and 1 ms. The fully reduced flavodoxin subsequently recombines with P700+ with a t1/2 of 330 ms. A corresponding flavodoxin semiquinone radical signal is readily observed in the dark by room temperature electron paramagnetic resonance, which reversibly disappears upon illumination. In contrast, the light-induced reduction of oxidized flavodoxin can be observed only by first-flash experiments following excessive dark adaptation. In addition, the docking site of flavodoxin on photosystem I was determined by electron microscopy in combination with image analysis. Flavodoxin binds to the cytoplasmic side of photosystem I at a distance of 7 nm from the centre of the trimer and in close contact to a ridge formed by the subunits PsaC, PsaD and PsaE.

Laser flash absorption spectroscopy study of flavodoxin reduction by photosystem I in Synechococcus sp. PCC 7002.

The photoreduction of flavodoxin by trimeric photosystem I, both from the cyanobacterium Synechococcus sp. PCC 7002, was investigated by flash absorption spectroscopy. After addition of flavodoxin in darkness, single flash experiments show that the transient signals change between individual flashes. This behavior is assigned to a progressive accumulation of flavodoxin semiquinone, which is relatively stable under most experimental conditions. Different conditions were devised in order to study the reduction of the oxidized and semiquinone forms of flavodoxin separately. Both processes were identified by their differential spectra measured between 460 and 630 nm. Detailed kinetic characteristics of flavodoxin reduction were obtained at pH 8.0 in the presence of salts. The kinetics of reduction of oxidized flavodoxin displays a single-exponential component. The rate of this component increases with the flavodoxin concentration up to an asymptotic value of about 600 s-1. The semiquinone form of flavodoxin being protonated, this rate corresponds to a rate-limiting reaction which could be either an electron transfer reaction or a protonation reaction. In contrast, the reduction of flavodoxin semiquinone is biphasic. A fast first-order phase with t 1/2 approximately 10 microseconds is interpreted as an electron transfer process within a preformed complex. A dissociation constant of 2.64 microM is calculated for this complex by assuming a simple binding equilibrium between photosystem I and flavodoxin semiquinone. The slower phase observed for semiquinone reduction is concentration dependent, and a second-order rate constant of 1.7 x 10(8) M-1 s-1 is calculated. For both one-electron reduction steps, different optimal salt concentrations are observed indicating slightly different interactions between photosystem I and flavodoxin in its oxidized and semiquinone states.

Interaction between photosystem I and flavodoxin from the cyanobacterium Synechococcus sp. PCC 7002 as revealed by chemical cross-linking.

The interaction between photosystem I (PS I) and flavodoxin from the cyanobacterium Synechococcus sp. PCC 7002 was investigated by covalent cross-linking in the presence of a hydrophilic cross-linker, N- ethyl-3-(3-diaminopropyl)carbodiimide. Under the experimental conditions employed, five distinct cross-linking products of flavodoxin and PS I subunits are formed. Immunoblot analyses show that these species are the result of cross-linking of flavodoxin to PsaC, PsaD, an unidentified low-molecular-mass PS I polypeptide, and a 15-kDa subunit. The latter has been indirectly identified as the PsaF subunit. Analysis of the interaction of flavodoxin with PS I from a psaE mutant indicates that the PsaE subunit is required for correct complex formation between flavodoxin and PS I, although this subunit is not directly cross-linked to flavodoxin. In addition, the cross-linking products of PsaD with PsaC and PsaL, and PsaE with PsaF, are observed. The covalent complex of flavodoxin and PS I is shown to be fully inhibited with respect to electron transfer to soluble flavodoxin, ferredoxin or ferredoxin:NADP+ oxidoreductase.

PsaE Is Required for in Vivo Cyclic Electron Flow around Photosystem I in the Cyanobacterium Synechococcus sp. PCC 7002.

Electron transfer rates to P700+ have been determined in wild-type and three interposon mutants (psaE-, ndhF-, and psaE- ndhF-) of Synechococcus sp. PCC 7002. All three mutants grew significantly more slowly than wild type at low light intensities, and each failed to grow photoheterotrophically in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and a metabolizable carbon source. The kinetics of P700+ reduction were similar in the wild-type and mutant whole cells in the absence of DCMU. In the presence of DCMU, the P700+ reduction rate in the psaE mutant was significantly slower than in the wild type. In the presence of DCMU and potassium cyanide, added to inhibit the outflow of electrons through cytochrome oxidase, P700+ reduction rates increased for both the psaE- and ndhF- strains. The reduction rates for these two mutants were nonetheless slower than that observed for the wild-type strain. The further addition of methyl viologen caused the rate of P700+ reduction in the wild type to become as slow as that for the psaE mutant in the absence of methyl viologen. Given the ability of methyl viologen to intercept electrons from the acceptor side of photosystem I, this response reveals a lesion in cyclic electron flow in the psaE mutant. In the presence of DCMU, the rate of P700+ reduction in the psaE ndhF double mutant was very slow and nearly identical with that for the wild-type strain in the presence of 2,4-dibromo-3-methyl-6-isopropyl-p-benzoquinone, a condition under which physiological electron donation to P700+ should be completely inhibited. These results suggest that NdhF- and PsaE-dependent electron donation to P700+ occurs only via plastoquinone and/or cytochrome b6/f and indicate that there are three major electron sources for P700+ reduction in this cyanobacterium. We conclude that, although PsaE is not required for linear electron flow to NADP+, it is an essential component in the cyclic electron transport pathway around photosystem I.

Cloning and characterization of the psaE gene of the cyanobacterium Synechococcus sp. PCC 7002: characterization of a psaE mutant and overproduction of the protein in Escherichia coli.

The psaE gene, encoding a 7.5 kDa peripheral protein of the photosystem I complex, has been cloned and characterized from the cyanobacterium Synechococcus sp. PCC 7002. The gene is transcribed as an abundant monocistronic transcript of approximately 325 nt. The PsaE protein has been overproduced in Escherichia coli, purified to homogeneity, and used to raise polyclonal antibodies. Mutant strains, in which the psaE gene was insertionally inactivated by interposon mutagenesis, were constructed and characterized. Although the PS I complexes of these strains were similar to those of the wild type, the strains grew more slowly under conditions which favour cyclic electron transport and could not grow at all under photoheterotrophic conditions. The results suggest that PsaE plays a role in cyclic electron transport in cyanobacteria.

Genes encoding eleven subunits of photosystem I from the thermophilic cyanobacterium Synechococcus sp.

We have isolated the genes encoding 11 photosystem I (PSI) subunits from Synechococcus sp., from which this reaction center has been crystallized. The recombinant DNAs, including psaA, psaB, psaC, psaD, psaE, psaF, psaI, psaJ, psaK and psaL, were obtained by heterologous hybridization with probes from appropriate cDNAs or genes from spinach and Synechocystis sp. PCC 6803, or with synthetic oligodeoxyribonucleotides. Genes psaA/psaB, psaF/psaJ and psaL/psaI are each closely linked. The open reading frames predict polypeptides of 83 kDa (subunits Ia and Ib, encoded by genes psaA and psaB, respectively), 15.4 kDa (II, psaD), 17.7 kDa (III, psaF), 8.4 kDa (IV, psaE), 8.8 kDa (VII, psaC), 4.6 kDa (VIII, psaI), 4.8 kDa (IX, psaJ), 8.5 kDa (X, psaK) and 15.5 kDa (XI, psaL). A novel subunit (XII, psaM) was also identified. Subunits II, III, IV and VII seem to be peripheral, while the others seem to be intrinsic components of the reaction center. These data imply a striking similarity of cyanobacterial and eukaryotic PSI. All subunits studied are encoded by single-copy genes which seem to be transcribed into monocistronic (psaC, psaD, psaC, psaK) or dicistronic (psaA/psaB, psaF/psaJ, psaL/psaI) RNA species. Subunit III is translated as a 17.7-kDa precursor, including a transit peptide of 23 amino acid residues. This is consistent with its location in the thylakoid lumen.