Spectroscopic study of the chromophore–protein association and primary photoinduced events in the photoreceptor of Blepharisma japonicum

Abstract

Blepharisma japonicum is a ciliated protozoan exhibiting a strong step-up photophobic response upon illumination. The photoreceptor chromophores responsible for this response have been identified to be hypericin-like chromophores (blepharismin and oxyblepharismin), complexed to a 200 kDa non-water-soluble protein. The present work opens up new perspectives on the primary phototransduction steps of B. japonicum's light perception through a joined approach by steady-state fluorescence spectroscopy, time-resolved fluorescence anisotropy and sub-picosecond transient absorption spectroscopy. The free chromophore of the light-adapted form of the cell (oxyblepharismin) was studied in various solvents and its spectroscopic properties, as well as its primary excited-state reactivity, compared with those of the corresponding pigment-protein complex, extracted by phosphate-concentration-step chromatography on a hydroxyapatite column. Fluorescence anisotropy together with SDS PAGE electrophoresis results confirm that oxyblepharismin is non-covalently bound to the apoprotein and show that, in the excited state, it is free to rotate in all directions within the binding site where it experiences a large local viscosity. Time-resolved anisotropy measurements on aromatic amino acids confirm that the molecular weight of the protein is of the order of 200 kDa. Although showing very similar steady-state spectra, free oxyblepharismin and its protein complex have noticeably different excited-state behaviours. In particular, the protein complex exhibits a pronounced short-lived absorption feature in the 640--750 nm range, decaying biexponentially in 4 ps and 56 ps. Those decays, also observed in other spectral regions, are not found in the corresponding kinetics of the isolated pigment in solution. This early behaviour of the protein complex might be the signature of the primary phototransduction process, possibly involving an electron transfer from the pigment to a neighbouring protein acceptor residue as it had been suggested in previous studies.

Photophysics and Multifunctionality of Hypericin‐Like Pigments in Heterotrich Ciliates: A Phylogenetic Perspective

“…In cases where a fluorescence quantum yield was not directly obtained, the value reported for a compound, i , is given by , where the subscript, hyp, denotes the quantities for hypericin in organic solvents such as DMSO. The directly and indirectly determined fluorescence quantum yield measurements do not agree for the oxyblepharismin/protein complex, which suggests that short lived lifetime components are not being detected in the measurement in reference (40). §See Figure 10 for more detail.…”

“…The photophobic behavior of B. japonicum is similar to that of S. coeruleus and the photoreceptor is blepharismin (73,89). However, rather than going into reverse, the cell pivots around the adoral side, making a 180° turn before moving forward (40,72). While Kraml and Marwan (72) concluded that B. japonicum lacked the negative phototaxis seen in S. coeruleus, Matsuoka (89) at the same time presented evidence for its occurrence.…”

“…Toxicity of blepharismin and stentorin chromophores in the light comes about primarily because they generate singlet molecular oxygen ( 1 O 2 ) and/or other reactive oxygen species, both in situ and in the medium (see section , above) (39,40,49,51). Surprisingly, some blepharismins and oxyblepharismins are also exogenously toxic in the dark to the predatory haptorid ciliate Dileptus margaritifer (42); this must be due to some other mechanism.…”

“…3) and Blepharisma japonicum these pigments are the photoreceptors for abrupt light‐avoidance responses. This role is well established, but there are few details of the signaling cascades leading to ciliary stroke reversal (35–40). These species are, however, susceptible to light in the presence of oxygen and must stay in dim light or darkness to survive endogenous toxicity of their own pigment (37,41,42).…”

Phosducin interacts with the G-protein βγ-dimer of ciliate protozoanBlepharisma japonicumupon illumination

“…Each granule contains the hipericin-like pigment blepharismin, which is thought to function as the primary photoreceptor eliciting the cell photoresponses (Tao et al, 1994;Song, 1997;Maeda et al, 1997;Matsuoka et al, 1997;Matsuoka et al, 2000). Recent analysis of primary photoprocesses in Blepharisma has revealed, however, that blepharismin undergoes an initial photocycle in the picosecond regime, indicating that the chromophore reaction in these cells does not play any active role in the phototransduction chain (Plaza et al, 2005;Plaza et al, 2007). The possibility that another class of photoreceptors may be utilized by Blepharisma in its photophobic behavior results also from our preliminary investigations, which showed the presence of a presumed rhodopsin-related protein in the cell membrane (H.F., unpublished data), as has been reported for other ciliates (Podesta et al, 1994;Nakaoka et al, 1991;Shinozawa et al, 1996).…”