Investigation of metabolic changes in living cells of the Chl d- containing cyanobacterium Acaryochloris marina by time- and wavelength correlated single-photon counting

Franz-Josef Schmitt ¹ , Christoph Theiss ¹ , Karin Wache ¹ , Justus Fuesers ¹ , Stefan Andree ¹ , Hans Joachim Eichler ¹ and Hann-Jörg Eckert 2

¹ Optisches Institut - P 1-1, Technische Universität Berlin, Strasse des 17. Juni 135, D-10623 Berlin, ² Max-Volmer-Laboratorium, Technische Universität Berlin, Strasse des 17. Juni 135, D-10623 Berlin

Content

1. Introduction S. 4

2. Materials and methods S. 6

3. Results S. 10

4. Decoupling of the PBPs under cold stress S. 14

5. Excited state populations according to a model of rate equations S. 18

6. Discussion S. 21

7. References S. 23

8. About the author S. 26

Abstract

Time- and wavelength-resolved fluorescence spectroscopy is an appropriate tool for quantitative and non-invasive investigations of living cells. Short measurement times with low excitation light intensities are necessary to observe variations of the fluorescence due to changes in the metabolism of the sensitive biological organisms. With new techniques the fluorescence dynamics can be monitored simultaneously in a broad spectrum during a very short measurement time. That provides information about the spectral differences of the fluorescence dynamics which can vary in correlation with the metabolic changes.

The interaction of the photosynthetic subunits and especially the mechanisms regulating the energy transfer are presently interesting and open fields in photosynthesis research. The phototrophic cyanobacterium Acaryochloris marina contains membrane extrinsic PBP antenna complexes and mainly Chl d containing membrane intrinsic core antenna complexes which absorb light and transfer excitation energy to the reaction center. The results of our studies suggest a fast excitation energy transfer kinetics of 20-30 ps along the PBP antenna of A.marina followed by a transfer with a time constant of about 60 ps to Chl d.

Very often cells or cell fragments are kept at low temperature to decelerate ageing processes. Living cells of A. marina which were stored at 0°C for some time showed a reduced excitation energy transfer from the PBP to the Chl d antenna, which partially recovered when the sample had been kept at 25 °C for a short time.

The reduction of the excitation energy transfer might be caused by a mechanism that decouples the PBP antenna under cold stress conditions avoiding photo damage of the reaction center of PS II.

Keywords: Acaryochloris marina, fluorescence dynamics, spectroscopy, phycobiliprotein, chlorophyll d, fluorescence lifetime, excitation energy transfer, phycocyanin, allophycocyanin, cell metabolism, photosynthesis research, biotechnology, biomedicine, life sciences

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Veränderungen im Metabolismus lebender Zellen des Chl d- haltigen Cyanobakteriums Acaryochloris marina untersucht mittels zeit- und wellenlängenkorrelierten Einzelphotonenzählens

Zusammenfassung

Zeit- und wellenlängenaufgelöste Fluoreszenzspektroskopie ist ein geeignetes Verfahren zur quantitativen und nichtinvasiven Untersuchung lebender Zellen. Kurze Messzeiten bei niedrigen Anregungsintensitäten sind notwendig, um Veränderungen der Fluoreszenz, verursacht durch metabolische Änderungen in empfindlichen biologischen Organismen, zu beobachten.

Mit moderner Technik kann die Fluoreszenzdynamik simultan in einem breiten Spektrum innerhalb einer kurzen Messzeit abgebildet werden. Dies liefert Informationen über spektrale Unterschiede der zeitabhängigen Fluoreszenz, welche in Korrelation zu metabolischen Veränderungen variieren kann.

Die Wechselwirkung der photosynthetischen Untereinheiten und insbesondere die Regulationsmechanismen des Energietransfers sind gegenwärtig interessante und offene Fragen der Photosyntheseforschung.

Das phototrophe Cyanobakterium Acaryochloris marina enthält membranextrinsische PBP Antennenkomplexe und überwiegend Chl d haltige membranintrinsische Core-Antennenkomplexe, welche Licht absorbieren und die Anregungsenergie zum Reaktionszentrum weiterleiten.

Die Ergebnisse unserer Untersuchungen weisen auf einen schnellen Energietransfer entlang der PBP Antenne mit einer Kinetik von 20-30 ps hin, auf welchen ein Transfer zum Chl d mit einer Zeitkonstanten von etwa 60 ps folgt.

Sehr oft werden Zellen oder Zellfragmente gekühlt, um Alterungsprozesse zu verlangsamen. Lebende Zellen von A. marina, die einige Zeit bei 0°C gelagert wurden, zeigten einen reduzierten Anregungsenergietransfer von der PBP Antenne zum Chl d, welcher sich innerhalb kurzer Zeit teilweise regenerierte, nachdem die Probe auf 25 °C erwärmt worden war.

Dieser Reduktion des Anregungsenergietransfers könnte ein Mechanismus zu Grunde liegen, welcher die PBP Antenne unter Kältestress entkoppelt, um eine lichtinduzierte Zerstörung des Reaktionszentrums im PS II zu verhindern.

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1. Introduction

Acaryochloris marina which was discovered in 1996 has a unique composition of the light harvesting system. The chlorophyll (Chl) antenna of Photosystem II (PS II) contains mainly Chl d instead of the usually dominant Chl a and the Phycobiliprotein (PBP) antenna has a simpler rod shaped structure than in typical cynobacteria [1]. The energy transfer processes are still not fully explained and further spectroscopic studies are necessary to answer the open questions.

During the past 20 years there has been a remarkable growth in the use of fluorescence techniques in biological sciences. Quantification of toxic substances or health promoting components in fruits and vegetables, environmental monitoring, clinical chemistry and DNA sequencing are just a few areas of application [2].

With fluorescence spectrometry the wavelength- and also the time-dependency of the emitted light can be used to identify and quantify fluorescent substances.

In photosynthesis research information on excitation energy transfer among antenna pigments, charge separation within the reaction centers, forming of channels for non-photochemical quenching and the pigment-pigment or pigment-protein coupling can be gathered from analyses of the time decay and wavelength dependence of the emitted fluorescence [3],[4]. In living cells changes in the fluorescence emission can be detected that correspond to the cell metabolism. The influence of stress factors like cold, heat, high light intensities or the starvation of essential components alters the conformation of the coupled pigment-protein complexes. In order to take full advantage of the information content of the fluorescence emission, it is necessary to monitor the time- and the wavelength dependency of fluorescence light with sufficient resolution because the broad fluorescence emission bands are the sum of the fluorescence of hundreds of pigments in different protein environments. Therefore the information about the electronic structure of a single pigment bound to a protein cannot be resolved. Mathematical data analysis and the theory of optical spectra which today delivers tools to calculate the absorption of pigment-protein complexes are helpful to extract information of specific molecules in a pool of fluorescing pigments [5]. When observing fast metabolic changes the signal has to be collected simultaneously in the time and wavelength domain, because the fluorescence decay at different wavelengths can only be compared if the time resolved fluorescence is measured at different wavelengths at the same time. Therefore fast acquisition of time resolved data in a broad spectral range is required which can be accomplished by new spectrometer systems.

In this study we use time- and wavelength-resolved single photon counting to investigate the excited states dynamics in living cells of the prokaryotic cyanobacterium Acaryochloris marina.

A.marina is a very special prokaryotic cyanobacterium. It is still the only known oxygenic photosynthetic organism containing Chl d as the dominant antenna pigment. Additionally Phycobiliproteins (PBPs) and small amounts of Chl a are present [1],[6]. Chl d has a formyl group on ring 1 of the porphyrin headgroup, in place of the vinyl group in Chl a, shifting the Q Y absorption maximum to 696 nm in methanol, ~30 nm more to the red as compared to Chl a (665 nm in methanol). A.marina is therefore able to exploit the near infrared light that penetrates to the shady environment where it lives [7]. In whole cells of A.marina, the main red absorption band is observed at 714-718 nm [1]. The room temperature steady state fluorescence of A. marina exhibits a broad Chl d band at 724 nm [8]. The red shift in

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absorbance of Chl d relative to Chl a is equal to an electronic energy gap of ~100 mV. Therefore it has been questioned how Chl d is able to split water in PS II [9],[10],[11]. Absorption- and fluorescence-spectroscopic studies with time and wavelength resolution have clarified the pathway of excitation energy transfer on the molecular level [11]. Also the mechanism of energy transfer and water splitting process has been analyzed in detail [12]. Chl d was shown to be the primary donor of the reaction center (RC) of PS I in A. marina [13]. The nature of the primary donor of PS II in A. marina is still in discussion. Recent studies suggest a start of the primary charge separation from the accessory Chl pigment which is Chl d (Chl d 1 ). This Chl d molecule is stabilized by the so-called special pair which consists of Chl a. Then the charge is quickly localized at Chl a. Therefore both Chlorophyll types, Chl a and Chl d are essential for the photochemistry in PS II of A.marina [11]. Fig. 1 shows a scheme of the cell and the PS II antenna system of A.marina. The prokaryotic cells of A.marina are containing staples of the thylakoid membrane where the photosynthesis takes place. The membrane extrinsic antenna is represented by a PBP rod which is associated to the PS II core antenna containing Chl d [14].

Fig. 1 Scheme of a cell of A.marina containing the thylakoid membrane (left side). At the right side the Light harvesting antenna complexes and Reaction center of PS II are shown according to Marquardt et al. [14]

The PBPs of A.marina have been reported to form aggregates of a simpler structure than those in typical cyanobacteria (fig. 1) [14]. They consist of four hexameric units, which resemble the peripheral rods of the typical cyanobacterial phycobilisomes (PBS) [14],[15]. Three of the hexamers were suggested to contain only phycocyanin (PC) and one to be a hetero-hexamer containing PC and allophycocyanin (APC). The excitation energy seems to be funneled directly from the APC-containing hetero-hexamer to Chl d of PS II without the involvement of an APC core as in typical cyanobacteria [15]. Isolated PBP aggregates of A. marina exhibit a fluorescence maximum at 665 nm (from APC) with a shoulder at about 655 nm (from PC) at room temperature [14]. This emission is also found in living cells of A.marina and the fluorescence decays with a time constant of 70 ps which is indicating the fast energy transfer from PBP to Chl d [16]. For reviews on fluorescence of A. marina and comparison with other systems, see Mimuro [17] and Itoh [18].

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