K.J. Hellingwerf

The Xanthopsins: a new family of eubacterial blue-light photoreceptors

Photoactive yellow protein (PYP) is a photoreceptor that has been isolated from three halophilic phototrophic purple bacteria. The PYP from Ectothiorhodospira halophila BN9626 is the only member for which the sequence has been reported at the DNA level. Here we describe the cloning and sequencing of the genes encoding the PYPs from E.halophila SL‐1 (type strain) and Rhodospirillum salexigens. The latter protein contains, like the E.halophila PYP, the chromophore trans p‐coumaric acid, as we show here with high performance capillary zone electrophoresis. Additionally, we present evidence for the presence of a gene encoding a PYP homolog in Rhodobacter sphaeroides, the first genetically well‐characterized bacterium in which this photoreceptor has been identified. An ORF downstream of the pyp gene from E.halophila encodes an enzyme, which is proposed to be involved in the biosynthesis of the chromophore of PYP. The pyp gene from E.halophila was used for heterologous overexpression in both Escherichia coli and R.sphaeroides, aimed at the development of a holoPYP overexpression system (an intact PYP, containing the p‐coumaric acid chromophore and displaying the 446 nm absorbance band). In both organisms the protein could be detected immunologically, but its yellow color was not observed. Molecular genetic construction of a histidine‐tagged version of PYP led to its 2500‐fold overproduction in E.coli and simplified purification of the heterologously produced apoprotein. HoloPYP could be reconstituted by the addition of p‐coumaric anhydride to the histidine‐tagged apoPYP (PYP lacking its chromophore). We propose to call the family of photoactive yellow proteins the xanthopsins, in analogy with the rhodopsins.

Ultrafast infrared spectroscopy reveals a key step for successful entry into the photocycle for photoactive yellow protein

Photoactive proteins such as PYP (photoactive yellow protein) are generally accepted as model systems for studying protein signal state formation. PYP is a blue-light sensor from the bacterium Halorhodospira halophila. The formation of PYPs signaling state is initiated by trans-cis isomerization of the p-coumaric acid chromophore upon the absorption of light. The quantum yield of signaling state formation is ≈0.3. Using femtosecond visible pump/mid-IR probe spectroscopy, we investigated the structure of the very short-lived ground state intermediate (GSI) that results from an unsuccessful attempt to enter the photocycle. This intermediate and the first stable GSI on pathway into the photocycle, I0, both have a mid-IR difference spectrum that is characteristic of a cis isomer, but only the I0 intermediate has a chromophore with a broken hydrogen bond with the backbone N atom of Cys-69. We suggest, therefore, that breaking this hydrogen bond is decisive for a successful entry into the photocycle. The chromophore also engages in a hydrogen-bonding network by means of its phenolate group with residues Tyr-42 and Glu-46. We have investigated the role of this hydrogen bond by exchanging the H bond-donating residue Glu-46 with the weaker H bond-donating glutamine (i.e., Gln-46). We have observed that this mutant exhibits virtually identical kinetics and product yields as WT PYP, even though during the I0-to-I1 transition, on the 800-ps time scale, the hydrogen bond of the chromophore with Gln-46 is broken, whereas this hydrogen bond remains intact with Glu-46.

Comparison of acid denaturation and light-activation in the eubacterial blue-light receptor photoactive yellow protein

Photoactive yellow protein PYP is a novel type of photoreceptor containing a thiol ester-linked p-coumarate anion chromophore. Photoexcitation of PYP triggers a photocycle which involves at least two intermediates: an early red-shifted . state and a long-lived blue-shifted state pB . At pH values below 3 PYP is reversibly converted into a stable blue-shifted . state pB . Here we quantify the transition from pG to pB at reduced pH as a two-state transition with an apparent p K dark dark of 2.8 and a steepness of 1.35 and report that the formation of pB is also induced by increased pressure midpoint dark . ; 1250 atm at pH 2.7 . The last step in the photocycle of PYP, from pB back to pG, is strongly decelerated by acidification. By global analysis of the data we calculated the UVrVis absorbance spectral and kinetic properties of pB and pB, dark together with their pH dependencies between pH 5 and 2. Similarities between pB and pB were found with respect to dark their absorbance spectra in both the UV and visible region and with respect to the effect of pH on their stability. It is proposed that an increase in acidity andror pressure leads to the steady state partial unfolding of PYP, while photoexcitia- tion leads to an analogous but transient unfolding process. q 1997 Elsevier Science B.V. .

Quantitative agreement between the values for the light-induced ΔpH in Rhodopseudomonas sphaeroides measured with automated flow-dialysis and 31P-NMR

Quantitative determination of transmembrane pH and electrical potential gradients is a prerequisite for a further refinement of the concepts of the chemiosmotic hypothesis [ 11. Several methods are available for the measurements of ApH and A

Sequence, chromophore extraction and 3-D model of the photoactive yellow protein from Rhodobacter sphaeroides

(review [21), of which the ‘spectroscopic’ and ‘distribution’ methods are most widely used. The outcome of the various methods, however, does show significant variations and it has been established by stringent comparisons, that the optical methods overestimate the magnitude of the transmembrane gradients [3-61. The distribution methods [2,7] (except flow-dialysis [S]) have the disadvantage that leakage of the probe molecules may occur during the separation step. Furthermore, in any distribution method uncertainties remain concerning: (i) The homogeneity of the internal aqueous phase and the absence of subcellular compartments; (ii) The ‘ideal behaviour’ [2] of the indicator probe; (iii) The activity coefficient of the probe molecules in the internal aqueous phase. With the application of 3’P NMR to biological systems [9-l l] a powerful and independent method has become available for the quantitation of ApH. This technique makes use of the pH dependence of 3’P NMR chemical shifts of phosphate metabolites. It can be used only if calibration curves of the chemical shift

From primary photochemistry to biological function in the blue-light photoreceptors PYP and AppA

The photoactive yellow protein (pyp) gene has been isolated from Rhodobacter sphaeroides by probing with a homologous PCR-product. A sequence analysis shows that this pyp gene encodes a 124 AA protein with 48% identity to the three known PYPs. Downstream from pyp, a number of adjacent open reading frames were identified, including a gene encoding a CoA-ligase homologue (pCL). This latter protein is proposed to be involved in PYP chromophore activation, required for attachment to the apoprotein. We have demonstrated the presence of the chromophoric group, previously identified in PYP from Ectothiorhodospira halophila as trans 4-hydroxy cinnamic acid, in phototrophically cultured R. sphaeroides cells by capillary zone electrophoresis. The basic structure of the chromophore binding pocket in PYP has been conserved, as shown by a 3D model of R. sphaeroides PYP, constructed by homology-based molecular modelling. In addition, this model shows that R. sphaeroides PYP contains a characteristic, positively charged patch.

The transmembrane electrical potential in intact bacteria: simultaneous measurements of carotenoid absorbance changes and lipophilic cation distribution in intact cells of Rhodobacter sphaeroides

To properly respond to changes in fluency conditions, Nature has developed a variety of photosensors that modulate gene expression, enzyme activity and/or motility. Dedicated types have evolved, which can be classified in six families: rhodopsins, phytochromes, xanthopsins, cryptochromes, phototropins and BLUF-proteins. The photochemistry of the first three families is based on cis/trans isomerization of an ethylene bond. Surprisingly, the latter three all use flavin as their chromophore, but each with very different photochemistry. In this contribution we will discuss the molecular basis of signal generation in a xanthopsin (Photoactive Yellow Protein (PYP) from Halorhodospira halophila), a photoreceptor for negative phototaxis, and in a BLUF protein (AppA from Rhodobacter sphaeroides), a transcriptional anti-repressor. PYP is activated through trans/cis isomerization of the 7,8-vinyl bond of its 4-hydroxycinnamic acid chromophore. This initiates a photocycle with multiple intermediates, like pB, which is formed after intramolecular proton transfer. The negative charge thus formed in the interior of the protein triggers formation of a partially unfolded signaling state. For AppA much less is known about the underlying photochemistry. Available evidence suggests that it is based on a light-induced change in the hydrogen-bonding of its flavin chromophore and/or a change in hydrophobic stacking between the flavin and/or nearby aromatic amino acids like Y 21. A signaling state is formed within microseconds, which recovers with a rate of approximately 10(-3) s(-1). The change in conformation between receptor- and signaling-state in AppA, however, appear to be minute as compared to those in PYP. Here we review the underlying chemistry in the various steps of the photocycle of these two photoreceptor proteins and provide new data on their mechanism and function.

31P NMR studies of photophosphorylation in intact cells of Chromatium vinosum

Abstract The electrical potential across the cytoplasmic membrane of Rhodobacter sphaeroides has been measured in intact cells by two independent techniques: the uptake and release of tetraphenyl phosphonium ions and the carotenoid absorbance band-shifts. Simultaneous measurements show that these two procedures give different membrane potentials. Upon energization with either light or during respiration tetraphenylphosphonium-distribution indicates a depolarization of the membrane while the electrochromic carotenoid band-shift indicates a hyperpolarisation. Treatment of the cells with venturicidin resulted in an increased light-induced membrane potential indicated by the carotenoid band-shift and led to a reversal in the polarity of the tetraphenylphosphonium response. The presence of ethylene diaminetetraacetic acid had no effect on the light-induced carotenoid absorbance change, but it decreased the light-induced membrane depolarisation indicated by the tetraphenylphosphonium ions. These results show that at least one of these methods is seriously in error.

A POLYVINYLCHLORIDE-MEMBRANE BASED ANION SELECTIVE ELECTRODE FOR CONTINUOUS REGISTRATION OF DELTA-PH (INTERIOR ALKALINE) WITH SALICYLATE AS THE INDICATOR PROBE

alp NMR contributed significantly to our understanding of the bioenergetics of intact cells and organs (review [1,2]). Being a non-invasive technique alp NMR allows direct determinations of the concentrations of phosphorylated metabolites within intact cells and tissues [3-5] and gives information about their cellular environment [6]. In addition it is extremely powerful in observing possible intracellular compartmentation [7-9] and in establishing heterogeneity of the cellular system, especially with respect to internal pH [10-12]. In [ 13,14], we reported the application of alp NMR to study energy transduction in Rhodopseudomonas sphaeroides, In this phototrophic purple nonsulfur bacterium an homogeneous intracellular compartment was revealed [13] and the magnitude of the light-induced transmembranal pH gradient could be quantitatively established [14]. No evidence for a distinct intrachromatophore compartment was obtained. In spite of the fact that adenine nucleotides could be demonstrated chemically [13], no ADP and A.TP resonances could be observed in alp NMR spectra of Rps. sphaeroides cells. Here we extend our studies to the phototrophic purple sulfur bacterium Chromatium vinosum. By extraction methods it has been shown that Chr. vinosum contains high levels of adenine nucleotides [ 15]. This bacterium is assumed to have a distinct intrachromatophore space, which is membrane-

Spectral identification of the electrochromically active carotenoids of Rhodobacter sphaeroides in chromatophores and reconstituted liposomes

An anion sensitive electrode has been constructed with the use of the lipid soluble cation benzyl-dimethyl-hexadecylammonium analogous to the procedure described for tetraphenylphosphonium-sensitive electrodes [Shinbo, T., Kamo, N., Kurihara, K. and Kobatake, Y. (1978) Arch. Biochem. Biophys. 187, 414-422]. The anion electrode responds to salicylate concentrations above 400 microM with a Nernstian sensitivity. Less lipid soluble anions like chloride and phosphate do not interfere. Below 400 microM salicylate the response of the electrode decreases gradually so that the sensitivity of the electrode is less than 10 mV per decade change at concentrations of the anion of 50 microM. A computer program has been developed to fit the electrode response curve with a polynomal function of the fourth power. Additional software-allows calculation of changes in the concentration of the salicylate anion, also under conditions where the sensitivity of the electrode for the anion is not constant. In this way the electrode can be used to measure changes in salicylate concentration that occur in a suspension of bacteria when, upon energization, a pH gradient is generated. 31P nuclear magnetic resonance measurements demonstrated that the pH gradient measured with the salicylate-sensitive electrode in the phototrophic bacterium Rhodopseudomonas sphaeroides is quantitatively correct. The response time of the electrode decreases from 1 min at 20 microM salicylate to 10 s at concentrations greater than or equal to 200 microM.