Mikhail L. Antonkine

Assembly of photosystem I. I. Inactivation of the rubA gene encoding a membrane-associated rubredoxin in the cyanobacterium Synechococcus sp. PCC 7002 causes a loss of photosystem I activity.

A 4.4-kb HindIII fragment, encoding an unusual rubredoxin (denoted RubA), a homolog of theSynechocystis sp. PCC 6803 gene slr2034 andArabidopsis thaliana HCF136, and the psbEFLJoperon, was cloned from the cyanobacterium Synechococcussp. PCC 7002. Inactivation of the slr2034 homolog produced a mutant with no detectable phenotype and wild-type photosystem (PS) II levels. Inactivation of the rubA gene ofSynechococcus sp. PCC 7002 produced a mutant unable to grow photoautotrophically. RubA and PS I electron transport activity were completely absent in the mutant, although PS II activity was ∼80% of the wild-type level. RubA contains a domain of ∼50 amino acids with very high similarity to the rubredoxins of anaerobic bacteria and archaea, but it also contains a region of about 50 amino acids that is predicted to form a flexible hinge and a transmembrane α-helix at its C terminus. Overproduction of the water-soluble rubredoxin domain inEscherichia coli led to a product with the absorption and EPR spectra of typical rubredoxins. RubA was present in thylakoid but not plasma membranes of cyanobacteria and in chloroplast thylakoids isolated from spinach and Chlamydomonas reinhardtii. Fractionation studies suggest that RubA might transiently associate with PS I monomers, but no evidence for an association with PS I trimers or PS II was observed. PS I levels were significantly lower than in the wild type (∼40%), but trimeric PS I complexes could be isolated from the rubA mutant. These PS I complexes completely lacked the stromal subunits PsaC, PsaD, and PsaE but contained all membrane-intrinsic subunits. The three missing proteins could be detected immunologically in whole cells, but their levels were greatly reduced, and degradation products were also detected. Our results indicate that RubA plays a specific role in the biogenesis of PS I.

Iron–sulfur clusters in type I reaction centers

Type I reaction centers (RCs) are multisubunit chlorophyll-protein complexes that function in photosynthetic organisms to convert photons to Gibbs free energy. The unique feature of Type I RCs is the presence of iron-sulfur clusters as electron transfer cofactors. Photosystem I (PS I) of oxygenic phototrophs is the best-studied Type I RC. It is comprised of an interpolypeptide [4Fe-4S] cluster, F(X), that bridges the PsaA and PsaB subunits, and two terminal [4Fe-4S] clusters, F(A) and F(B), that are bound to the PsaC subunit. In this review, we provide an update on the structure and function of the bound iron-sulfur clusters in Type I RCs. The first new development in this area is the identification of F(A) as the cluster proximal to F(X) and the resolution of the electron transfer sequence as F(X)-->F(A)-->F(B)-->soluble ferredoxin. The second new development is the determination of the three-dimensional NMR solution structure of unbound PsaC and localization of the equal- and mixed-valence pairs in F(A)(-) and F(B)(-). We provide a survey of the EPR properties and spectra of the iron-sulfur clusters in Type I RCs of cyanobacteria, green sulfur bacteria, and heliobacteria, and we summarize new information about the kinetics of back-reactions involving the iron-sulfur clusters.

Assembly of Photosystem I II. RUBREDOXIN IS REQUIRED FOR THE IN VIVO ASSEMBLY OF FX IN SYNECHOCOCCUS SP. PCC 7002 AS SHOWN BY OPTICAL AND EPR SPECTROSCOPY

The rubA gene was insertionally inactivated in Synechococcus sp. PCC 7002, and the properties of photosystem I complexes were characterized spectroscopically. X-band EPR spectroscopy at low temperature shows that the three terminal iron-sulfur clusters, FX, FA, and FB, are missing in whole cells, thylakoids, and photosystem (PS) I complexes of the rubAmutant. The flash-induced decay kinetics of both P700+ in the visible and A1 − in the near-UV show that charge recombination occurs between P700+ and A1 − in both thylakoids and PS I complexes. The spin-polarized EPR signal at room temperature from PS I complexes also indicates that forward electron transfer does not occur beyond A1. In agreement, the spin-polarized X-band EPR spectrum of P700+ A1 − at low temperature shows that an electron cycle between A1 − and P700+ occurs in a much larger fraction of PS I complexes than in the wild-type, wherein a relatively large fraction of the electrons promoted are irreversibly transferred to [FA/FB]. The electron spin polarization pattern shows that the orientation of phylloquinone in the PS I complexes is identical to that of the wild type, and out-of-phase, spin-echo modulation spectroscopy shows the same P700+ to A1 − center-to-center distance in photosystem I complexes of wild type and the rubA mutant. In contrast to the loss of FX, FB, and FA, the Rieske iron-sulfur protein and the non-heme iron in photosystem II are intact. It is proposed that rubredoxin is specifically required for the assembly of the FX iron-sulfur cluster but that FX is not required for the biosynthesis of trimeric P700-A1 cores. Since the PsaC protein requires the presence of FX for binding, the absence of FA and FB may be an indirect result of the absence of FX.

Synthesis and characterization of de novo designed peptides modelling the binding sites of [4Fe-4S] clusters in photosystem I.

Photosystem I (PS I) converts the energy of light into chemical energy via transmembrane charge separation. The terminal electron transfer cofactors in PS I are three low-potential [4Fe-4S] clusters named F(X), F(A) and F(B), the last two are bound by the PsaC subunit. We have modelled the F(A) and F(B) binding sites by preparing two apo-peptides (maquettes), sixteen amino acids each. These model peptides incorporate the consensus [4Fe-4S] binding motif along with amino acids from the immediate environment of the iron-sulfur clusters F(A) and F(B). The [4Fe-4S] clusters were successfully incorporated into these model peptides, as shown by optical absorbance, EPR and Mössbauer spectroscopies. The oxidation-reduction potential of the iron-sulfur cluster in the F(A)-maquette is -0.44+/-0.03 V and in the F(B)-maquette is -0.47+/-0.03 V. Both are close to that of F(A) and F(B) in PS I and are considerably more negative than that observed for other [4Fe-4S] model systems described earlier (Gibney, B. R., Mulholland, S. E., Rabanal, F., and Dutton, P. L. Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 15041-15046). Our optical data show that both maquettes can irreversibly bind to PS I complexes, where PsaC-bound F(A) and F(B) were removed, and possibly participate in the light-induced electron transfer reaction in PS I.

Solution structure of the unbound, oxidized Photosystem I subunit PsaC, containing [4Fe-4S] clusters F A and F B : a conformational change occurs upon binding to Photosystem I

Abstract. This work presents the three-dimensional NMR solution structure of recombinant, oxidized, unbound PsaC from Synechococcus sp. PCC 7002. Constraints are derived from homo- and heteronuclear one-, two- and three-dimensional 1H and 15N NMR data. Significant differences are outlined between the unbound PsaC structure presented here and the available X-ray structure of bound PsaC as an integral part of the whole cyanobacterial PS I complex. These differences mainly concern the arrangement of the N- and C-termini with respect to the [4Fe-4S] core domain. In the NMR solution structure of PsaC the C-terminal region assumes a disordered helical conformation, and is clearly different from the extended coil conformation, which is one of the structural elements required to anchor PsaC to the PS I core heterodimer. In solution the N-terminus of PsaC is in contact with the pre-C-terminal region but slides in between the latter and the iron-sulfur core region of the protein. Together, these features result in a concerted movement of the N-terminus and pre-C-terminal region away from the FA binding site, accompanied by a bending of the N-terminus. In comparison, the same terminal regions are positioned much closer to FA and take up an anti-parallel β-sheet arrangement in PsaC bound to PS I. The conformational changes between bound and unbound PsaC correlate with the differences reported earlier for the EPR spectra of reduced FA and FB in bound versus unbound PsaC. The observed different structural features in solution are highly relevant for unraveling the stepwise assembly process of the stromal PsaC, PsaD and PsaE subunits to the PS I core heterodimer. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0321-3.

Electronic Structure of the Quinone Radical Anion A1˙― of Photosystem I Investigated by Advanced Pulse EPR and ENDOR Techniques

Vitamin K1 (VK1) is an important cofactor of the electron-transfer chain in photosystem I (PS I), referred to as A1. The special properties of this quinone result from its unique interaction(s) with the protein surrounding. In particular, a single H-bond to neutral A1 was identified previously in the X-ray crystal structure of PS I. During light-induced electron transfer in PS I, A1 is transiently reduced to the radical anion A1*-. In this work, we characterized the electron spin density distribution of A1*- with the aim of understanding the influence of the protein surrounding on it. We studied the light-induced spin-polarized radical pair P700*+A1*- and the photoaccumulated radical anion A1*-, using advanced pulse EPR, ENDOR, and TRIPLE techniques at Q-band (34 GHz). Exchange with fully deuterated quinone in the A1 binding site allowed differentiation between proton hyperfine couplings from the quinone and from the protein surrounding. In addition, DFT calculations on a model of the A1 site were performed and provided proton hyperfine couplings that were in close agreement with the ones determined experimentally. This combined approach allowed the assignment of proton hyperfine coupling tensors to molecular positions, thereby yielding a picture of the spin density distribution in A1*-. Comparison with VK1*- in organic solvents (Epel et al. J. Phys. Chem. B 2006, 110, 11549.) leads to the conclusion that the single H-bond present in both the radical pair P700*+A1*- and the photoaccumulated radical anion A1*- is, indeed, the crucial factor that governs the electronic structure of A1*-.

Paramagnetic 1H NMR spectroscopy of the reduced, unbound Photosystem I subunit PsaC: sequence-specific assignment of contact-shifted resonances and identification of mixed-and equal-valence Fe-Fe pairs in [4Fe-4S] centers FA− and FB−

The PsaC subunit of Photosystem I (PS I) is a 9.3-kDa protein that binds two important cofactors in photosynthetic electron transfer: the [4Fe-4S] clusters FA and FB. The g-tensor orientation of FA− and FB− is believed to be correlated to the preferential localization of the mixed-valence and equal-valence (ferrous) iron pairs in each [4Fe-4S]+ cluster. The preferential position of the mixed-valence and equal-valence pairs, in turn, can be inferred from the study of the temperature dependence of contactshifted resonances by 1H NMR spectroscopy. For this, a sequence-specific assignment of these signals is required. The 1H NMR spectrum of reduced, unbound PsaC from Synechococcus sp. PCC 7002 at 280.4 K in 99% D2O solution shows 18 hyperfine-shifted resonances. The non-solvent-exchangeable, hyperfineshifted resonances of reduced PsaC are clearly identified as belonging to the cysteines coordinating the clusters FA− and FB− by their downfield chemical shifts, by their temperature dependencies, and by their short T1 relaxation times. The usual fast method of assigning the 1H NMR spectra of reduced [4Fe-4S] proteins through magnetization transfer from the oxidized to the reduced state was not feasible in the case of reduced PsaC. Therefore, a de novo self-consistent sequence-specific assignment of the hyperfine-shifted resonances was obtained based on dipolar connectivities from 1D NOE difference spectra and on longitudinal relaxation times using the X-ray structure of Clostridium acidi urici 2[4Fe-4S] cluster ferredoxin at 0.94 Å resolution as a model. The results clearly show the same sequence-specific distribution of Curie and anti-Curie cysteines for unbound, reduced PsaC as established for other [4Fe-4S]-containing proteins; therefore, the mixed-valence and equal-valence (ferrous) Fe-Fe pairs in FA− and FB− have the same preferential positions relative to the protein. The analysis reveals that the magnetic properties of the two [4Fe-4S] clusters are essentially indistinguishable in unbound PsaC, in contrast to the PsaC that is bound as a component of the PS I complex.

Chemical Rescue of Site-Modified Ligands to the Iron-Sulfur Clusters of Psac In Photosystem I

The PsaC subunit of Photosystem I (PS I) is an 8.6 kDa protein which contains binding sites for two [4Fe-4S] clusters. To investigate structure-function relationships in PsaC we changed cysteine 14, which ligates FB, to glycine [1]. After cluster reconstitution unbound PsaC showed an EPR spectrum consistent with the presence of two [4Fe-4S] clusters, one S = 1/2 and the other S = 3/2. We proposed that 2-mercaptoethanol, a reagent used in the reconstitution protocol, serves as an external rescue ligand in the absence of a biological ligand to the fourth iron [1]. Instead of the expected formation of a [3Fe-4S] cluster in the glycine mutants, chemical rescue allows for the formation of a [4Fe-4S] cluster. The present work is aimed at obtaining direct evidence to support the chemical rescue hypothesis. We reconstituted the C14G/C34S mutant of PsaC with 4-fluorothiophenol or 2,2,2-tlifluoroethanethiol as the external thiolate ligand. Successful insertion of [4Fe-4S] clusters was confirmed by electron paramagnetic resonance (EPR) spectroscopy. Flourine-19 nuclear magnetic resonance (19F NMR) spectroscopy showed paramagnetically shifted resonances that were attributed to cluster-bound ligand.

De novo Peptides Modeling the Binding Sites of [4Fe-4S] Clusters in Photosystem I

Photosystem I (PSI) converts the energy of light into chemical energy. The terminal electron transfer cofactors in PS I are three iron-sulfur clusters named FX, FA and FB. The PsaC subunit of PS I harbors binding sites of the [4Fe-4S] clusters FA and FB. We modeled them by preparing two peptides (maquettes), sixteen amino acids each, using Fmoc solid state peptide synthesis. These model peptides incorporate the consensus iron-sulfur binding motif along with amino acids from the immediate environment of the respective iron-sulfur cluster. The [4Fe-4S] clusters were successfully incorporated into these model peptides, as shown by their optical absorbance, EPR and Mossbauer spectra. The oxidation- reduction potential of the iron-sulfur clusters in the model peptides is close to that of FA and FB in PsaC at room temperature and is considerably lower than that observed for other [4Fe-4S] model systems described earlier.

A Rubredoxin-Like Protein Plays an Essential Role in Assembly of the F A , F B & F X Iron-Sulfur Clusters in Photosystem I

The photosystem I (PS I) reaction center is a large multisubunit complex composed of at least eleven polypeptides in cyanobacteria and chloroplasts of higher plants [1]. Six redox centers are involved in light-induced electron transfer from plastocyanin (or cytochrome c6) to ferredoxin (or flavodoxin) in PS I. The PsaA/PsaB heterodimer harbors the primary electron carriers P700 (a chlorophyll (Chl) a dimer), A0 (a monomeric Chl a), A1 (a phylloquinone) and FX (an interpolypeptide [4Fe–4S] cluster). The terminal electron acceptors FA and FB are [4Fe–4S] clusters which are located on the extrinsic PsaC protein. Although significant progress has been achieved in elucidating the structure [2, 3] and function of the PS I, questions still remain concerning the biogenesis, assembly, and regulation of PS I in the membrane. The biogenesis of photosynthetic complexes in cyanobacteria and higher-plant chloroplasts is probably a complex, multi-step process, which is likely to be highly regulated at the post-translation level, especially for cofactor-binding polypeptides. Here we report preliminary results from studies of a gene encoding a novel rubredoxin-like protein with an essential function in the assembly of the FA, FB and FX iron-sulfur clusters in PS I reaction center.