Confinement in crystal lattice alters entire photocycle pathway of the Photoactive Yellow Protein.

Femtosecond time-resolved crystallography (TRC) on proteins enables resolving the spatial structure of short-lived photocycle intermediates. An open question is whether confinement and lower hydration of the proteins in the crystalline state affect the light-induced structural transformations. Here, we measured the full photocycle dynamics of a signal transduction protein often used as model system in TRC, Photoactive Yellow Protein (PYP), in the crystalline state and compared those to the dynamics in solution, utilizing electronic and vibrational transient absorption measurements from 100 fs over 12 decades in time. We find that the photocycle kinetics and structural dynamics of PYP in the crystalline form deviate from those in solution from the very first steps following photon absorption. This illustrates that ultrafast TRC results cannot be uncritically extrapolated to in vivo function, and that comparative spectroscopic experiments on proteins in crystalline and solution states can help identify structural intermediates under native conditions.

Functional Expression of Gloeobacter Rhodopsin in PSI-Less Synechocystis sp. PCC6803.

The approach of providing an oxygenic photosynthetic organism with a cyclic electron transfer system, i.e., a far-red light-driven proton pump, is widely proposed to maximize photosynthetic efficiency via expanding the absorption spectrum of photosynthetically active radiation. As a first step in this approach, Gloeobacter rhodopsin was expressed in a PSI-deletion strain of Synechocystis sp. PCC6803. Functional expression of Gloeobacter rhodopsin, in contrast to Proteorhodopsin, did not stimulate the rate of photoheterotrophic growth of this Synechocystis strain, analyzed with growth rate measurements and competition experiments. Nevertheless, analysis of oxygen uptake and-production rates of the Gloeobacter rhodopsin-expressing strains, relative to the ΔPSI control strain, confirm that the proton-pumping Gloeobacter rhodopsin provides the cells with additional capacity to generate proton motive force. Significantly, expression of the Gloeobacter rhodopsin did modulate levels of pigment formation in the transgenic strain.

Combining retinal-based and chlorophyll-based (oxygenic) photosynthesis: Proteorhodopsin expression increases growth rate and fitness of a ∆PSI strain of Synechocystis sp. PCC6803.

To fill the "green absorption gap", a green absorbing proteorhodopsin was expressed in a PSI-deletion strain (ΔPSI) of Synechocystis sp. PCC6803. Growth-rate measurements, competition experiments and physiological characterization of the proteorhodopsin-expressing strains, relative to the ΔPSI control strain, allow us to conclude that proteorhodopsin can enhance the rate of photoheterotrophic growth of ΔPSI Synechocystis strain. The physiological characterization included measurement of the amount of residual glucose in the spent medium and analysis of oxygen uptake- and production rates. To explore the use of solar radiation beyond the PAR region, a red-shifted variant Proteorhodopsin-D212N/F234S was expressed in a retinal-deficient PSI-deletion strain (ΔPSI/ΔSynACO). Via exogenous addition of retinal analogue an infrared absorbing pigment (maximally at 740 nm) was reconstituted in vivo. However, upon illumination with 746 nm light, it did not significantly stimulate the growth (rate) of this mutant. The inability of the proteorhodopsin-expressing ΔPSI strain to grow photoautotrophically is most likely due to a kinetic rather than a thermodynamic limitation of its NADPH-dehydrogenase in NADP+-reduction.

Deletion of sll1541 in Synechocystis sp. Strain PCC 6803 Allows Formation of a Far-Red-Shifted holo-Proteorhodopsin In Vivo.

In many pro- and eukaryotes, a retinal-based proton pump equips the cell to drive ATP synthesis with (sun)light. Such pumps, therefore, have been proposed as a plug-in for cyanobacteria to artificially increase the efficiency of oxygenic photosynthesis. However, little information on the metabolism of retinal, their chromophore, is available for these organisms. We have studied the in vivo roles of five genes (sll1541, slr1648, slr0091, slr1192, and slr0574) potentially involved in retinal metabolism in Synechocystis sp. strain PCC 6803. With a gene deletion approach, we have shown that Synechocystis apo-carotenoid-15,15-oxygenase (SynACO), encoded by gene sll1541, is an indispensable enzyme for retinal synthesis in Synechocystis, presumably via asymmetric cleavage of ß-apo-carotenal. The second carotenoid oxygenase (SynDiox2), encoded by gene slr1648, competes with SynACO for substrate(s) but only measurably contributes to retinal biosynthesis in stationary phase via an as-yet-unknown mechanism. In vivo degradation of retinal may proceed through spontaneous chemical oxidation and via enzyme-catalyzed processes. Deletion of gene slr0574 (encoding CYP120A1), but not of slr0091 or of slr1192, causes an increase (relative to the level in wild-type Synechocystis) in the retinal content in both the linear and stationary growth phases. These results suggest that CYP120A1 does contribute to retinal degradation. Preliminary data obtained using 13C-labeled retinal suggest that conversion to retinol and retinoic acid and subsequent further oxidation also play a role. Deletion of sll1541 leads to deficiency in retinal synthesis and allows the in vivo reconstitution of far-red-absorbing holo-proteorhodopsin with exogenous retinal analogues, as demonstrated here for all-trans 3,4-dehydroretinal and 3-methylamino-16-nor-1,2,3,4-didehydroretinal.IMPORTANCE Retinal is formed by many cyanobacteria and has a critical role in most forms of life for processes such as photoreception, growth, and stress survival. However, the metabolic pathways in cyanobacteria for synthesis and degradation of retinal are poorly understood. In this paper we identify genes involved in its synthesis, characterize their role, and provide an initial characterization of the pathway of its degradation. This led to the identification of sll1541 (encoding SynACO) as the essential gene for retinal synthesis. Multiple pathways for retinal degradation presumably exist. These results have allowed us to construct a strain that expresses a light-dependent proton pump with an action spectrum extending beyond 700 nm. The availability of this strain will be important for further work aimed at increasing the overall efficiency of oxygenic photosynthesis.

Functional Expression of Gloeobacter Rhodopsin in Synechocystis sp. PCC6803.

Proteorhodopsins are light-driven proton pumps that occur widespread in Nature, where they function predominantly in environments with high incident irradiance. Their maximal absorbance is usually in the blue range, but can be extended into the (far)red range of the electromagnetic spectrum. Because they can be expressed heterologously, they may be exploited in studies aimed at increasing the efficiency of photosynthesis. Here we report further studies toward this goal, by comparing the expression of two different bacterial rhodopsins (Proteorhodopsin and Gloeobacter rhodopsin) in the model cyanobacterium Synechocystis sp. PCC6803. In particular, we investigated the pigments bound by the respective apo-opsins, and the oligomeric state of the corresponding holo-rhodopsins, both in Escherichia coli and in the cyanobacterial membranes. We conclude that the two proton-pumping rhodopsins are predominantly present in an oligomeric state (hexamers for Proteorhodopsin and trimers for Gloeobacter rhodopsin). Furthermore, Gloeobacter rhodopsin is able to bind an antenna carotenoid (in addition to retinal) and has the highest pumping rate at given light intensity. However, its lower expression level will decrease its physiological effectiveness. It remains to be established which of these two bacterial rhodopsins is best in stimulating the growth rate of its cyanobacterial host.

Short hydrogen bonds and negative charge in photoactive yellow protein promote fast isomerization but not high quantum yield.

Biological signal transduction by photoactive yellow protein (PYP) in halophilic purple sulfur bacteria is initiated by trans-to-cis isomerization of the p-coumaric acid chromophore (pCa) of PYP. pCa is engaged in two short hydrogen bonds with protein residues E46 and Y42, and it is negatively charged at the phenolate oxygen. We investigated the role in the isomerization process of the E46 short hydrogen bond and that of the negative charge on the anionic phenolate moiety of the chromophore. We used wild-type PYP and the mutant E46A, in protonated and deprotonated states (referred to as pE46A and dpE46A, respectively), to reduce the number of hydrogen bond interactions between the pCa phenolate oxygen and the protein and to vary the negative charge density in the chromophore-binding pocket. Their effects on the yield and rate of chromophore isomerization were determined by ultrafast spectroscopy. Molecular dynamics simulations were used to relate these results to structural changes in the mutant protein. We found that deprotonated pCa in E46A has a slower isomerization rate as the main part of this reaction was associated with time constants of 1 and 6 ps, significantly slower than the 0.6 ps time constant in wild-type PYP. The quantum yield of isomerization in dpE46A was estimated to be 30 ± 2%, and that of pE46A was 32 ± 3%, very close to the value determined for wtPYP of 32 ± 2%. Relaxation of the isomerized product state I0 to I1 was faster in dpE46A. We conclude that the negative charge on pCa stabilized by the short hydrogen bonds with E46 and Y42 affects the rate of isomerization but not the quantum yield of isomerization. With this information, we propose a scheme for the potential energy surfaces involved in the isomerization and suggest protein motions near the pCa backbone as key events in successful isomerization.

H(2)O(2) production in species of the Lactobacillus acidophilus group: a central role for a novel NADH-dependent flavin reductase.

Hydrogen peroxide production is a well-known trait of many bacterial species associated with the human body. In the presence of oxygen, the probiotic lactic acid bacterium Lactobacillus johnsonii NCC 533 excretes up to 1 mM H(2)O(2), inducing growth stagnation and cell death. Disruption of genes commonly assumed to be involved in H(2)O(2) production (e.g., pyruvate oxidase, NADH oxidase, and lactate oxidase) did not affect this. Here we describe the purification of a novel NADH-dependent flavin reductase encoded by two highly similar genes (LJ_0548 and LJ_0549) that are conserved in lactobacilli belonging to the Lactobacillus acidophilus group. The genes are predicted to encode two 20-kDa proteins containing flavin mononucleotide (FMN) reductase conserved domains. Reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin and is specific for NADH and not NADPH. The Km for FMN is 30 ± 8 µM, in accordance with its proposed in vivo role in H(2)O(2) production. Deletion of the encoding genes in L. johnsonii led to a 40-fold reduction of hydrogen peroxide formation. H(2)O(2) production in this mutant could only be restored by in trans complementation of both genes. Our work identifies a novel, conserved NADH-dependent flavin reductase that is prominently involved in H(2)O(2) production in L. johnsonii.

Photoionization and electron radical recombination dynamics in photoactive yellow protein investigated by ultrafast spectroscopy in the visible and near-infrared spectral region.

Photoinduced ionization of the chromophore inside photoactive yellow protein (PYP) was investigated by ultrafast spectroscopy in the visible and near-infrared spectral regions. An absorption band that extended from around 550 to 850 nm was observed and ascribed to solvated electrons, ejected from the p-hydroxycinnamic acid anion chromophore upon the absorption of two 400 nm photons. Global kinetic analysis showed that the solvated electron absorption decayed in two stages: a shorter phase of around 10 ps and a longer phase of more than 3 ns. From a simulation based on a diffusion model we conclude that the diffusion rate of the electron is about 0.8 Å(2)/ps in wild type PYP, and that the electron is ejected to a short distance of only several angstroms away from the chromophore. The chromophore-protein pocket appears to provide a water-similar local environment for the electron. Because mutations at different places around the chromophore have different effect on the electron recombination dynamics, we suggest that solvated electrons could provide a new method to investigate the local dielectric environment inside PYP and thus help to understand the role of the protein in the photoisomerization process.

Selective enrichment and identification of cross-linked peptides to study 3-D structures of protein complexes by mass spectrometry.

Chemical cross-linking of protein complexes combined with mass spectrometry is a powerful approach to obtain 3-D structural information by revealing amino residues that are in close spatial proximity. To increase the efficiency of mass spectrometric analysis, we have demonstrated the selective enrichment of cross-linked peptides from the 350 kDa protein complex RNA polymerase (RNAP) from Bacillus subtilis. Bis(succinimidyl)-3-azidomethyl glutarate was used as a cross-linker along with an azide-reactive cyclooctyne-conjugated resin to capture target peptides. Subsequently released peptides were fractionated by strong cation exchange chromatography and subjected to LC-MS/MS. We mapped 10 different intersubunit and 24 intrasubunit cross-links by xComb database searching supplied with stringent criteria for confirmation of the proposed structure of candidate cross-linked peptides. The cross-links fit into a homology model of RNAP. Cross-links between ß lobe 1 and the ß' downstream jaw, and cross-links involving the N-terminal and C-terminal parts of the α subunits suggest conformational flexibility. The analytical strategy presented here can be applied to map protein-protein interactions at the amino acid level in biological assemblies of similar complexity. Our approach enables the exploration of alternative peptide fragmentation techniques that may further facilitate cross-link analysis.

On the midpoint potential of the FAD chromophore in a BLUF-domain containing photoreceptor protein.

The redox-midpoint potential of the FAD chromophore in the BLUF domain of anti-transcriptional regulator AppA from Rhodobacter sphaeroides equals ∼-260mV relative to the calomel electrode. Altering the structure of its chromophore-binding pocket through site-directed mutagenesis brings this midpoint potential closer to that of free flavin in aqueous solution. The redox-midpoint potential of this BLUF domain is intermediate between those of LOV domains and Cryptochromes, which may rationalize the primary photochemistry observed in these three flavin-containing photoreceptor families. These results also imply that LOV domains, among the flavin-containing photosensory receptors, are least sensitive to intracellular chemical reduction in the dark.

Reaction pathways of photoexcited retinal in proteorhodopsin studied by pump-dump-probe spectroscopy.

Proteorhodopsin (pR) is a membrane-embedded proton pump from the microbial rhodopsin family. Light absorption by its retinal chromophore initiates a photocycle, driven by trans/cis isomerization on the femtosecond to picosecond time scales. Here, we report a study on the photoisomerization dynamics of the retinal chromophore of pR, using dispersed ultrafast pump-dump-probe spectroscopy. The application of a pump pulse initiates the photocycle, and with an appropriately tuned dump pulse applied at a time delay after the dump, the molecules in the initial stages of the photochemical process can be de-excited and driven back to the ground state. In this way, we were able to resolve an intermediate on the electronic ground state that represents chromophores that are unsuccessful in isomerization. In particular, the fractions of molecules that undergo slow isomerization (20 ps) have a high probability to enter this state rather than the isomerized K-state. On the ground state reaction surface, return to the stable ground state conformation via a structural or vibrational relaxation occurs in 2-3 ps. Inclusion of this intermediate in the kinetic scheme led to more consistent spectra of the retinal-excited state, and to a more accurate estimation of the quantum yield of isomerization (Phi = 0.4 at pH 6).

Characterization of the primary photochemistry of proteorhodopsin with femtosecond spectroscopy.

Proteorhodopsin is an ion-translocating member of the microbial rhodopsin family. Light absorption by its retinal chromophore initiates a photocycle, driven by trans/cis isomerization, leading to transmembrane translocation of a proton toward the extracellular side of the cytoplasmic membrane. Here we report a study on the photoisomerization dynamics of the retinal chromophore of proteorhodopsin, using femtosecond time-resolved spectroscopy, by probing in the visible- and in the midinfrared spectral regions. Experiments were performed both at pH 9.5 (a physiologically relevant pH value in which the primary proton acceptor of the protonated Schiff base, Asp(97), is deprotonated) and at pH 6.5 (with Asp(97) protonated). Simultaneous analysis of the data sets recorded in the two spectral regions and at both pH values reveals a multiexponential excited state decay, with time constants of approximately 0.2 ps, approximately 2 ps, and approximately 20 ps. From the difference spectra associated with these dynamics, we conclude that there are two chromophore-isomerization pathways that lead to the K-state: one with an effective rate of approximately (2 ps)(-1) and the other with a rate of approximately (20 ps)(-1). At high pH, both pathways are equally effective, with an estimated quantum yield for K-formation of approximately 0.7. At pH 6.5, the slower pathway is less productive, which results in an isomerization quantum yield of 0.5. We further observe an ultrafast response of residue Asp(227), which forms part of the counterion complex, corresponding to a strengthening of its hydrogen bond with the Schiff base on K-state formation; and a feature that develops on the 0.2 ps and 2 ps timescale and probably reflects a response of an amide II band in reaction to the isomerization process.

Binding, tuning and mechanical function of the 4-hydroxy-cinnamic acid chromophore in photoactive yellow protein.

The bacterial photoreceptor protein photoactive yellow protein (PYP) covalently binds the chromophore 4-hydroxy coumaric acid, tuning (spectral) characteristics of this cofactor. Here, we study this binding and tuning using a combination of pointmutations and chromophore analogs. In all photosensor proteins studied to date the covalent linkage of the chromophore to the apoprotein is dispensable for light-induced catalytic activation. We analyzed the functional importance of the covalent linkage using an isosteric chromophore-protein variant in which the cysteine is replaced by a glycine residue and the chromophore by thiomethyl-p-coumaric acid (TMpCA). The model compound TMpCA is shown to weakly complex with the C69G protein. This non-covalent binding results in considerable tuning of both the pKa and the color of the chromophore. The photoactivity of this system, however, was strongly impaired, making PYP the first known photosensor protein in which the covalent linkage of the chromophore is of paramount importance for the functional activity of the protein in vitro. We also studied the influence of chromophore analogs on the color and photocycle of PYP, not only in WT, but especially in the E46Q mutant, to test if effects from both chromophore and protein modifications are additive. When the E46Q protein binds the sinapinic acid chromophore, the color of the protein is effectively changed from yellow to orange. The altered charge distribution in this protein also results in a changed pKa value for chromophore protonation, and a strongly impaired photocycle. Both findings extend our knowledge of the photochemistry of PYP for signal generation.

A base-catalyzed mechanism for dark state recovery in the Avena sativa phototropin-1 LOV2 domain.

Phototropins are autophosphorylating serine/threonine kinases responsible for blue-light perception in plants; their action gives rise to phototropism, chloroplast relocation, and opening of stomatal guard cells. The kinase domain constitutes the C-terminal part of Avena sativa phototropin 1. The N-terminal part contains two light, oxygen, or voltage (LOV) sensing domains, LOV1 and LOV2; each binds a flavin mononucleotide (FMN) chromophore (lambdamax = 447 nm, termed D447) and forms the light-sensitive domains, of which LOV2 is the principal component. Blue-light absorption produces a covalent adduct between a very conserved nearby cysteine residue and the C(4a) atom of the FMN moiety via the triplet state of the flavin. The covalent adduct thermally decays to regenerate the D447 dark state, with a rate that may vary by several orders of magnitude between different species. We report that the imidazole base can act as a very efficient enhancer of the dark recovery of A. sativa phot1 LOV2 (AsLOV2) and some other well-characterized LOV domains. Imidazole accelerates the thermal decay of AsLOV2 by 3 orders of magnitude in the submolar concentration range, via a base-catalyzed mechanism involving base abstraction of the FMN N(5)-H adduct state and subsequent reprotonation of the reactive cysteine. The LOV2 crystal structure suggests that the imidazole molecules may act from a cavity located in the vicinity of the FMN, explaining its high efficiency, populated through a channel connecting the cavity to the protein surface. Use of pH titration and chemical inactivation by diethyl pyrocarbonate (DEPC) suggests that histidines located at the surface of the LOV domain act as base catalysts via an as yet unidentified H-bond network, operating at a rate of (55 s)-1 at pH 8. In addition, molecular processes other than histidine-mediated base catalysis contibute significantly to the total thermal decay rate of the adduct and operate at a rate constant of (65 s)-1, leading to a net adduct decay time constant of 30 s at pH 8.

Investigation of in vivo cross-talk between key two-component systems of Escherichia coli.

Intracellular signal transfer in bacteria is dominated by phosphoryl transfer between conserved transmitter and receiver domains in regulatory proteins of so-called two-component systems. Escherichia coli contains 30 such systems, which allow it to modulate gene expression, enzyme activity and the direction of flagellar rotation. The authors have investigated whether, and to what extent, these separate systems form (an) interacting network(s) in vivo, focussing on interactions between four major systems, involved in the responses to the availability of phosphorylated sugars (Uhp), phosphate (Pho), nitrogen (Ntr) and oxygen (Arc). Significant cross-talk was not detectable in wild-type cells. Decreasing expression levels of succinate dehydrogenase (reporting Arc activation), upon activation of the Pho system, appeared to be independent of signalling through PhoR. Cross-talk towards NtrC did occur, however, in a ntrB deletion strain, upon joint activation of Pho, Ntr and Uhp. UhpT expression was demonstrated when cells were grown on pyruvate, through non-cognate phosphorylation of UhpA by acetyl phosphate.