The homodimeric type I reaction center in heliobacteria is arguably the simplest known pigment–protein complex capable of conducting (bacterio)chlorophyll-based conversion of light into chemical energy. Despite its structural simplicity, the thermodynamics of the electron transfer cofactors on the acceptor side have not been fully investigated. In this work, we measured the midpoint potential of the terminal [4Fe-4S]2+/1+ cluster (FX) in reaction centers from Heliobacterium modesticaldum. The FX cluster was titrated chemically and monitored by (i) the decrease in the level of stable P800 photobleaching by optical spectroscopy, (ii) the loss of the light-induced g ≈ 2 radical from P800+• following a single-turnover flash, (iii) the increase in the low-field resonance at 140 mT attributed to the S = 3/2 ground spin state of FX–, and (iv) the loss of the spin-correlated P800+ FX– radical pair following a single-turnover flash. These four techniques led to similar estimations of the midpoint potential for FX of −502 ± 3 mV (n = 0.99), −496 ± 2 mV (n = 0.99), −517 ± 10 mV (n = 0.65), and −501 ± 4 mV (n = 0.84), respectively, with a consensus value of −504 ± 10 mV (converging to n = 1). Under conditions in which FX is reduced, the long-lived (∼15 ms) P800+ FX– state is replaced by a rapidly recombining (∼15 ns) P800+A0– state, as shown by ultrafast optical experiments. There was no evidence of the presence of a P800+ A1– spin-correlated radical pair by electron paramagnetic resonance (EPR) under these conditions. The midpoint potentials of the two [4Fe-4S]2+/1+ clusters in the low-molecular mass ferredoxins were found to be −480 ± 11 mV/–524 ± 13 mV for PshBI, −453 ± 6 mV/–527 ± 6 mV for PshBII, and −452 ± 5 mV/–533 ± 8 mV for HM1_2505 as determined by EPR spectroscopy. FX is therefore suitably poised to reduce one [4Fe-4S]2+/1+ cluster in these mobile electron carriers. Using the measured midpoint potential of FX and a quasi-equilibrium model of charge recombination, the midpoint potential of A0 was estimated to be −854 mV at room temperature. The midpoint potentials of A0 and FX are therefore 150–200 mV less reducing than their respective counterparts in Photosystem I of cyanobacteria and plants. This places the redox potential of the FX cluster in heliobacteria approximately equipotential to the highest-potential iron–sulfur cluster (FA) in Photosystem I, consistent with its assignment as the terminal electron acceptor.

•We express an active [FeFe] hydrogenase in the chloroplast of Chlamydomonas reinhardtii.•The chloroplast hydrogenase is no longer regulated by the native control system.•Production of the apoprotein occurs under both aerobic and anaerobic conditions.•Unregulated expression results in a strong negative selective pressure.•A vitamin-controlled gene repression system suitably manages the negative effects.Biological hydrogen generation from phototrophic organisms is a promising source of renewable fuel. The nuclear-expressed [FeFe] hydrogenase from Chlamydomonas reinhardtii has an extremely high turnover rate, and so has been a target of intense research. Here, we demonstrate that a codon-optimized native hydrogenase can be successfully expressed in the chloroplast. We also demonstrate a curiously strong negative selective pressure resulting from unregulated hydrogenase expression in this location, and discuss management of its expression with a vitamin-controlled gene repression system. To the best of our knowledge, this represents the first example of a nuclear-expressed, chloroplast-localized metalloprotein being synthesized in situ. Control of this process opens up several bioengineering possibilities for the production of biohydrogen.

Heliobacteria contain a very simple photosynthetic apparatus, consisting of a homodimeric type I reaction center (RC) without a peripheral antenna system and using the unique pigment bacteriochlorophyll (BChl) g. They are thought to use a light-driven cyclic electron transport pathway to pump protons, and thereby phosphorylate ADP, although some of the details of this cycle are yet to be worked out. We previously reported that the fluorescence emission from the heliobacterial RC in vivo was increased by exposure to actinic light, although this variable fluorescence phenomenon exhibited very different characteristics to that in oxygenic phototrophs (Collins et al. 2010). Here, we describe the underlying mechanism behind the variable fluorescence in heliobacterial cells. We find that the ability to stably photobleach P800, the primary donor of the RC, using brief flashes is inversely correlated to the variable fluorescence. Using pump-probe spectroscopy in the nanosecond timescale, we found that illumination of cells with bright light for a few seconds put them in a state in which a significant fraction of the RCs underwent charge recombination from P800+A0− with a time constant of ~20 ns. The fraction of RCs in the rapidly back-reacting state correlated very well with the variable fluorescence, indicating that nearly all of the increase in fluorescence could be explained by charge recombination of P800+A0−, some of which regenerated the singlet excited state. This hypothesis was tested directly by time-resolved fluorescence studies in the ps and ns timescales. The major decay component in whole cells had a 20-ps decay time, representing trapping by the RC. Treatment of cells with dithionite resulted in the appearance of a ~18-ns decay component, which accounted for ~0.6 % of the decay, but was almost undetectable in the untreated cells. We conclude that strong illumination of heliobacterial cells can result in saturation of the electron acceptor pool, leading to reduction of the acceptor side of the RC and the creation of a back-reacting RC state that gives rise to delayed fluorescence.

Cytochrome c553 of Heliobacterium modesticaldum is the donor to P800+, the primary electron donor of the heliobacterial reaction center (HbRC). It is a membrane-anchored 14-kDa cytochrome that accomplishes electron transfer from the cytochrome bc complex to the HbRC. The petJ gene encoding cyt c553 was cloned and expressed in Escherichia coli with a hexahistidine tag replacing the lipid attachment site to create a soluble donor that could be made in a preparative scale. The recombinant cytochrome had spectral characteristics typical of a c-type cytochrome, including an asymmetric α-band, and a slightly red-shifted Soret band when reduced. The EPR spectrum of the oxidized protein was characteristic of a low-spin cytochrome. The midpoint potential of the recombinant cytochrome was +217 ± 10 mV. The interaction between soluble recombinant cytochrome c553 and the HbRC was also studied. Re-reduction of photooxidized P800+ was accelerated by addition of reduced cytochrome c553. The kinetics were characteristic of a bimolecular reaction with a second order rate of 1.53 × 104 M−1 s−1 at room temperature. The rate manifested a steep temperature dependence, with a calculated activation energy of 91 kJ mol−1, similar to that of the native protein in Heliobacillus gestii cells. These data demonstrate that the recombinant soluble cytochrome is comparable to the native protein, and likely lacks a discrete electrostatic binding site on the HbRC.

A time-resolved spectroscopic study of the isolated photosynthetic reaction center (RC) from Heliobacterium modesticaldum reveals that thermal equilibration of light excitation among the antenna pigments followed by trapping of excitation and the formation of the charge-separated state P800+A0– occurs within ~25 ps. This time scale is similar to that reported for plant and cyanobacterial photosystem I (PS I) complexes. Subsequent electron transfer from the primary electron acceptor A0 occurs with a lifetime of ~600 ps, suggesting that the RC of H. modesticaldum is functionally similar to that of Heliobacillus mobilis and Heliobacterium chlorum. The (A0– − A0) and (P800+ − P800) absorption difference spectra imply that an 81-OH-Chl aF molecule serves as the primary electron acceptor and occupies the position analogous to ec3 (A0) in PS I, while a monomeric BChl g pigment occupies the position analogous to ec2 (accessory Chl). The presence of an intense photobleaching band at 790 nm in the (A0– − A0) spectrum suggests that the excitonic coupling between the monomeric accessory BChl g and the 81-OH-Chl aF in the heliobacterial RC is significantly stronger than the excitonic coupling between the equivalent pigments in PS I.

We have developed a purification protocol for photoactive reaction centers (HbRC) from Heliobacterium modesticaldum. HbRCs were purified from solubilized membranes in two sequential chromatographic steps, resulting in the isolation of a fraction containing a single polypeptide, which was identified as PshA by LC–MS/MS of tryptic peptides. All polypeptides reported earlier as unknown proteins (in Heinnickel et al., Biochemistry 45:6756–6764, 2006; Romberger et al., Photosynth Res 104:293–303, 2010) are now identified by mass spectrometry to be the membrane-bound cytochrome c553 and four different ABC-type transporters. The purified PshA homodimer binds the following pigments: 20 bacteriochlorophyll (BChl) g, two BChl g′, two 81-OH-Chl aF, and one 4,4′-diaponeurosporene. It lacks the PshB polypeptide binding the FA and FB [4Fe–4S] clusters. It is active in charge separation and exhibits a trapping time of 23 ps, as judged by time-resolved fluorescence studies. The charge recombination rate of the P800+FX− state is 10–15 ms, as seen before. The purified HbRC core was able to reduce cyanobacterial flavodoxin in the light, exhibiting a KM of 10 μM and a kcat of 9.5 s−1 under near-saturating light. There are ~1.6 menaquinones per HbRC in the purified complex. Illumination of frozen HbRC in the presence of dithionite can cause creation of a radical at g = 2.0046, but this is not a semiquinone. Furthermore, we show that high-purity HbRCs are very stable in anoxic conditions and even remain active in the presence of oxygen under low light.

The phylloquinone acceptor PhQA in photosystem I binds to the protein through a single H-bond to the backbone nitrogen of PsaA-L722. Here, we investigate the effect of this H-bond on the electron transfer (ET) kinetics by substituting threonine for PsaA-L722. Room temperature spin-polarized transient EPR measurements show that in the PsaA-L722T mutant, the rate of PhQA– to FX ET increases and the hyperfine coupling to the 2-methyl group of PhQA is much larger than in the wild type. Molecular dynamics simulations and ONIOM type electronic structure calculations indicate that it is possible for the OH group of the Thr side chain to form an H-bond to the carbonyl oxygen atom, O4 of the phylloquinone, and that this results in an increase in the 2-methyl hyperfine couplings as observed in the transient EPR data. The Arrhenius plot of the PhQA– to FX ET in the PsaA-L722T mutant suggests that the increased rate is probably the result of a slight change in the electronic coupling between PhQA– and FX. The strong deviation from Arrhenius behavior observed at ∼200 K can be reproduced using a semiclassical model, which takes the zero-point energy of the mode coupled to the ET into account. However, since the change in slope of the Arrhenius plot occurs at the protein glass transition temperature, it is argued that it could be the result of a change in the protein relaxation dynamics at this temperature rather than quantum mechanical effects.

In Photosystem 1 (PS1), phylloquinone (PhQ) acts as a secondary electron acceptor from chlorophyll ec3 and also as an electron donor to the iron–sulfur cluster FX. PS1 possesses two virtually equivalent branches of electron transfer (ET) cofactors from P700 to FX, and the lifetime of the semiquinone intermediate displays biphasic kinetics, reflecting ET along the two different branches. PhQ in PS1 serves only as an intermediate in ET and is not normally fully reduced to the quinol form. This is in contrast to PS2, in which plastoquinone (PQ) is doubly reduced to plastoquinol (PQH2) as the terminal electron acceptor. We purified PS1 particles from the menD1 mutant of Chlamydomonas reinhardtii that cannot synthesize PhQ, resulting in replacement of PhQ by PQ in the quinone-binding pocket. The magnitude of the stable flash-induced P700+ signal of menD1 PS1, but not wild-type PS1, decreased during a train of laser flashes, as it was replaced by a ∼30 ns back-reaction from the preceding radical pair (P700+A0–). We show that this process of photoinactivation is due to double reduction of PQ in the menD1 PS1 and have characterized the process. It is accelerated at lower pH, consistent with a rate-limiting protonation step. Moreover, a point mutation (PsaA-L722T) in the PhQA site that accelerates ET to FX ∼2-fold, likely by weakening the sole H-bond to PhQA, also accelerates the photoinactivation process. The addition of exogenous PhQ can restore activity to photoinactivated PS1 and confer resistance to further photoinactivation. This process also occurs with PS1 purified from the menB PhQ biosynthesis mutant of Synechocystis PCC 6803, demonstrating that it is a general phenomenon in both prokaryotic and eukaryotic PS1.

Photosystem I (PSI) is a large pigment-protein complex that unites a reaction center (RC) at the core with ∼100 core antenna chlorophylls surrounding it. The RC is composed of two cofactor branches related by a pseudo-C2 symmetry axis. The ultimate electron donor, P700 (a pair of chlorophylls), and the tertiary acceptor, FX (a Fe4S4 cluster), are both located on this axis, while each of the two branches is made up of a pair of chlorophylls (ec2 and ec3) and a phylloquinone (PhQ). Based on the observed biphasic reduction of FX, it has been suggested that both branches in PSI are competent for electron transfer (ET), but the nature and rate of the initial electron transfer steps have not been established. We report an ultrafast transient absorption study of Chlamydomonas reinhardtii mutants in which specific amino acids donating H-bonds to the 131-keto oxygen of either ec3A (PsaA-Tyr696) or ec3B (PsaB-Tyr676) are converted to Phe, thus breaking the H-bond to a specific ec3 cofactor. We find that the rate of primary charge separation (CS) is lowered in both mutants, providing direct evidence that the primary ET event can be initiated independently in each branch. Furthermore, the data provide further support for the previously published model in which the initial CS event occurs within an ec2/ec3 pair, generating a primary ec2+ec3- radical pair, followed by rapid reduction by P700 in the second ET step. A unique kinetic modeling approach allows estimation of the individual ET rates within the two cofactor branches.

The kinetics of electron transfer from phyllosemiquinone (PhQ•−) to the iron sulfur cluster FX in Photosystem I (PS I) are described by lifetimes of ∼20 and ∼250 ns. These two rates are attributed to reactions involving the quinones bound primarily by the PsaB (PhQB) and PsaA (PhQA) subunits, respectively. The factors leading to a ∼10-fold difference between the observed lifetimes are not yet clear. The peptide nitrogen of conserved residues PsaA-Leu722 and PsaB-Leu706 is involved in asymmetric hydrogen-bonding to PhQA and PhQB, respectively. Upon mutation of these residues in PS I of the green alga, Chlamydomonas reinhardtii, we observe an acceleration of the oxidation kinetics of the PhQ•− interacting with the targeted residue: from ∼255 to ∼180 ns in PsaA-L722Y/T and from ∼24 to ∼10 ns in PsaB-L706Y. The acceleration of the kinetics in the mutants is consistent with a perturbation of the H-bond, destabilizing the PhQ•− state, and increasing the driving force of its oxidation. Surprisingly, the relative amplitudes of the phases reflecting PhQA•− and PhQB•− oxidation were also affected by these mutations: the apparent PhQA•−/PhQB•− ratio is shifted from 0.65:0.35 in wild-type reaction centers to 0.5:0.5 in PsaA-L722Y/T and to 0.8:0.2 in PsaB-L706Y. The most consistent account for all these observations involves considering reversibility of oxidation of PhQA•− and PhQB•− by FX, and asymmetry in the driving forces for these electron transfer reactions, which in turn leads to Fx-mediated interquinone electron transfer.

Two functional electron transfer (ET) chains, related by a pseudo-C2 symmetry, are present in the reaction center of photosystem I (PSI). Due to slight differences in the environment around the cofactors of the two branches, there are differences in both the kinetics of ET and the proportion of ET that occurs on the two branches. The strongest evidence that this is indeed the case relied on the observation that the oxidation rates of the reduced phylloquinone (PhQ) cofactor differ by an order of magnitude. Site-directed mutagenesis of residues involved in the respective PhQ-binding sites resulted in a specific alteration of the rates of semiquinone oxidation. Here, we show that the PsaA-F689N mutation results in an ∼100-fold decrease in the observed rate of PhQA− oxidation. This is the largest change of PhQA− oxidation kinetics observed so far for a single-point mutation, resulting in a lifetime that exceeds that of the terminal electron donor, P700+. This situation allows a second photochemical charge separation event to be initiated before PhQA− has decayed, thereby mimicking in PSI a situation that occurs in type II reaction centers. The results indicate that the presence of PhQA− does not impact the overall quantum yield and leads to an almost complete redistribution of the fractional utilization of the two functional ET chains, in favor of the one that does not bear the charged species. The evolutionary implications of these results are also briefly discussed.