Target Analysis of Primary Photoprocesses Involved in the

Aug 1, 2017 - oxyblepharismin-binding protein (OBIP) thought to trigger the photophobic response of the ciliate Blepharisma japonicum. The OBIP sample...
0 downloads 0 Views 175KB Size
Download PDF
690

J. Phys. Chem. B 2007, 111, 690-696

Target Analysis of Primary Photoprocesses Involved in the Oxyblepharismin-Binding Protein Pascal Plaza,* Mathilde Mahet,† and Monique M. Martin De´ partement de Chimie, UMR 8640 CNRS-ENS, Ecole Normale Supe´ rieure, 24 rue Lhomond, 75005 Paris, France

Giovanni Checcucci and Francesco Lenci Istituto di Biofisica, Via G. Moruzzi 1, 56100 Pisa, Italy ReceiVed: July 6, 2006; In Final Form: NoVember 9, 2006

Target analysis is performed on previously published transient absorption spectra of the 200-kDa oxyblepharismin-binding protein (OBIP) thought to trigger the photophobic response of the ciliate Blepharisma japonicum. The OBIP sample is considered as heterogeneous and made of two distinct classes of chromophoreprotein complexes. A so-called nonreactive class is seen to be comparable to free oxyblepharismin in organic solution. Another, reactive, class is shown to undergo a fast picosecond photocycle involving the formation in 4 ps of an intermediate state noted Y1. The spectrum associated to Y1 bears striking similarities with that of the oxyblepharismin radical cation. This element favors the hypothesis that an excited-state intermolecular electron-transfer could be the primary step of the sensory transduction chain of B. japonicum. Proton release is also considered as a possible secondary step. These possibilities support the idea that reactive OBIP functions like an electron or proton pump. We alternatively propose a new hypothesis stating that the fast photocycle of reactive OBIP actually does not generate any photoproduct or protein change of conformation but is involved in another biological function. It would act as a kind of solar screen, providing additional protection to the light-adapted form of B. japonicum in case of excessive illumination.

1. Introduction Blepharisma japonicum is a red-colored ciliated protozoan that exhibits a strong step-up photophobic response.1,2 If red B. japonicum cells are continuously irradiated with weak intensities (3-30 W‚m-2), they photoconvert into blue, light-adapted, cells that still display the same phobic response.3 Blue cells are particularly interesting because a readily extractable and stable chromophore-protein complex has been proposed by Matsuoka to mediate their photophobic response.4,5 It is made of a nonsoluble 200-kDa protein, noncovalently6 bound to a chromophore called oxyblepharismin (OxyBP). For brevity, we shall refer to this complex as OBIP (oxyblepharismin-binding protein). Oxyblepharismin is the photooxidized form of blepharismin (BP), the photoreceptor molecule of red cells7-9 (Chart 1). The sequence, hence the three-dimensional (3D) structure, of OBIP is still unknown. Different schemes were proposed to describe the primary processes B. japonicum’s photosensory transduction chain.10-13 One of the most established steps is the occurrence of a photoinduced pH jump, observed by means of various techniques and characterized by an acidification of the intracellular medium.10,14,15 It was even proposed that protons are being translocated from granules containing the photoreceptor toward the cytoplasm of the cell.14 This fact suggests that a proton is likely to be released by the excited chromophore, as it has been * Corresponding author. Phone: +33 144322414. E-mail: Pascal.Plaza@ ens.fr. † Present address: Laboratoire d’Electrochimie Mole ´ culaire, UMR 7591 CNRS-Universite´ Paris 7, 2 place Jussieu, Tour 44-45, 4e Et., 75251 Paris Cedex 05, France.

CHART 1: Structures of Blepharismin (BP) and Oxyblepharismin (OxyBP)

proposed for the photoreceptor of another ciliate, Stentor coeruleus.16 It was later shown that photoinduced electron transfer from the first singlet excited state to a suitable electron acceptor was an efficient quenching mechanism of the fluorescence of free OxyBP in organic solution.12 It was suggested that such an electron transfer could also occur in vivo, a disulphide bridge playing the role of the electron acceptor.13 Proton release was supposed to easily follow this initial step due to an expected pronounced decrease of the pKa of OxyBP in its radical cation form, as is the case for tyrosine17 and was proposed for hypericin.18 In a preceding series of papers,6,19,20 we reported subpicosecond transient absorption experiments on the OBIP chromoprotein and showed that its behavior is quite different from that of free OxyBP in organic solution. While free OxyBP is mainly characterized by a 1.1-ns decay, up to 50% of OBIP’s excited-state decay is biexponential. The two components have 4- and 56-ps lifetimes, and their weights are roughly comparable. It is important to note that a bandlike structure centered around 680 nm was observed at the earliest times of the experiment

10.1021/jp0642591 CCC: $37.00 © 2007 American Chemical Society Published on Web 01/05/2007

Oxyblepharismin-Binding Protein Photoprocesses

J. Phys. Chem. B, Vol. 111, No. 4, 2007 691 spectra of OxyBP, both dissolved in DMSO and complexed with human serum albumin (HSA).20 This latter artificial complex was studied in order to approach the question of the specificity of the interaction between OxyBP and its native protein partner.20 We chose HSA because it is one of the most important blood proteins and because a similar complex with the analogous chromophore hypericin had already been prepared22-26 and studied by picosecond transient absorption spectroscopy.27 2. Methods

Figure 1. DADS corresponding to the 4-ps, 56-ps, and 1.8-ns lifetimes of OBIP.

SCHEME 1: Two-Population Kinetic Model Describing the Photoinduced Reactivity of OBIPa

a Here, 60% of the initial excited population is in X1 (rOBIP), and 40%, in Z1 (nrOBIP). 40% of the X1 population decays to Y1.

and seen to vanish in the hundred-picosecond time scale. The longer times are strikingly dominated by a transient species the spectrum of which is very similar to that of free OxyBP in solution. These facts were first tentatively interpreted by an excited-state photodissociation mechanism, but this hypothesis was ruled out by energy arguments based on the small steadystate Stokes shift of the OBIP system.20 We then reached the conclusion that OxyBP is heterogeneously bound to its protein partner, leading to the existence of a reactive and a nonreactive population. The reactive population gives rise to the specific spectral features, in particular in the 640-740 nm region, and to the fast 4- and 56-ps dynamics. The nonreactive population mostly behaves like the free chromophore in solution. We finally showed that the distinctive 680-nm band bears a close similarity with the spectrum of the OxyBP radical cation,20 which is an important element in favor of the electron-transfer hypothesis mentioned above. This paper is devoted to a target analysis21 of formerly published transient absorption spectra on OBIP, the time resolution of which was ca. 1.3 ps.6 This new analysis goes further than the global analysis performed at selected wavelengths which we recently reported.20 On the one hand, the full spectrum is now taken into account. More importantly, this new treatment produces a quantitative description of the spectra in terms of a comprehensive kinetic model, with rate constants and branching ratios, and characterizes each intermediate by its species-associated difference spectrum (SADS). Only such a target analysis can provide these valuable and new elements. The picture emerging from the present analysis is used to reexamine the nature of the primary photoprocesses at work in OBIP. Special care is devoted to the hypothesis of the photoinduced intermolecular electron transfer previously considered, but a new and important alternative is considered, suggesting a novel physiological role of the pigment. For the sake of comparison, the same type of analysis has been performed on also previously published transient absorption

Target analysis21 of the subpicosecond difference absorption data reported in references6,20 has been performed in two steps. The complete set of experimental data was first globally fitted to a sum of N exponential functions. Two methods were used to achieve this goal. The first one is based on the singular value decomposition (SVD)28 of the matrix (A) containing the twoway data (wavelength and time). In order to favorize the contribution of spectral regions displaying a large signal-tonoise (S/N) ratio, we weighted the data prior to the decomposition. We chose a difference spectrum at a given short time as the weighting spectrum. This weighting was subsequently reversed to obtain the final decomposition of A into exponential decays and decay-associated difference spectra (DADS). The other method consisted of getting the N decays by a global fit restricted to a limited number (typically 20) of significant wavelengths and subsequently calculating the DADS at all wavelengths by multiplying A by the Moore-Penrose inverse29 of the matrix representing those decays. We verified that both methods give very similar results. In the second step a kinetic model involving N transient species was chosen, the parameters of which were adjusted to be compatible with the N exponentials of the first step. This model allowed the calculation of the kinetics of the various transient species and their species-associated difference spectra (SADS).30 One should note that the parameters of the kinetic model may have been incompletely determined by the constraint on the exponential lifetimes. Since the models we chose were quite simple, we used an elementary method to adjust the remaining parameters: we imposed that all SADS exhibit approximately the same negative bleaching contribution. The meaning is simply that any transient state contributes in the same way to the bleaching of the initial ground state. We called this condition the “bleaching constraint”. As a heterogeneous model, involving two classes of molecules, was used for OBIP, the above constraint only applies under the assumption that the ground-state spectra attached to the two classes are strictly identical. Such an assumption is made throughout the following section. 3. Results and Discussion 3.1. The OBIP Chromoprotein. 3.1.1. Target Analysis. A previous multiexponential global fit at 15 selected wavelengths across the transient absorption spectra of OBIP in a phosphate buffer exhibited three exponential decay components, the lifetimes of which are 4 ps, 56 ps, and 1.8 ns.6 These lifetimes were used to obtain a full global fit at all recorded wavelengths by the Moore-Penrose method sketched above. Figure 1 shows the three corresponding DADS. On the basis of the qualitative analysis developed in ref 20 and recalled in the Introduction, the transient absorption spectra of OBIP are best understood as arising from a heterogeneous partition of the sample into a reactive population (noted rOBIP), characterized by specific spectral features in the 640-740-nm region and by the fast 4- and 56-ps dynamics, and a nonreactive

692 J. Phys. Chem. B, Vol. 111, No. 4, 2007

Figure 2. SADS of species X1, Y1, and Z1 from the two-population kinetic model of OBIP shown in Scheme 1.

Figure 3. Comparison of the transient absorption spectrum of free OxyBP in DMSO, for a pump-probe delay of 500 ps (blue), and the Z1 SADS of OBIP (green) considered in Scheme 1.

population (nrOBIP), mostly behaving like the free chromophore in solution. This representation was used to build the kinetic model of Scheme 1 where a fraction x of the initial excitedstate population is found in state X1, standing for the excited state of rOBIP, and (1 - x) is found in state Z1, representing the excited state of nrOBIP. State X1 decays in 4 ps and produces, with a yield R, state Y1 which returns to the ground state in 56 ps. State Z1 deactivates to the ground state in 1.8 ns. Using the bleaching constraint, the free parameters were adjusted to x ) 0.6 and R ) 0.4, with a relative error of about (15%. This means that 60% of the initial excited-state population is in the X1 reactive state and that 40% of it gives rise to state Y1 while 60% returns to the ground state. The direct ground-state recovery from X1 explains why no delayed rise of the transient absorption signal is observed around 680 nm in spite of the formation of Y1, which has a larger absorbance than X1 at this wavelength. Figure 2 shows the SADS of species X1, Y1, and Z1. The rate constant for the X1 f Y1 reaction was found to be 1011 s-1. It is worth noting that in such a simple scheme SADS(X1), SADS(Y1), and SADS(Z1) are respectively proportional to DADS1 + DADS2, DADS2, and DADS3. As Figure 3 shows, it should be noted that the SADS of Z1 is very similar to the transient absorption spectra of free OxyBP in solution. The observed slight shift of the negative bleaching and stimulated contributions of the spectra was expected since the steady-state absorption and fluorescence spectra of OBIP and free OxyBP are shifted from each other by a few

Plaza et al.

Figure 4. Comparison of the SADS of species Y1 (red) with the difference absorption spectrum assigned to OxyBP•+ (blue).

nanometers.20 Once this phenomenon has been taken into account, their global overlap is quite striking. It can thus be thought that Z1 corresponds to the excited state of a chromoprotein where the interaction between OxyBP and the apoprotein is weak. In addition, Figure 2 shows that the SADS of X1 and Y1 are globally similar, except for a much more pronounced peak at 680 nm for species Y1. This is quite intriguing because the 680nm band had been considered, at a qualitative level of analysis,6,20 as the specific signature of the rOBIP, apparently already present at the earliest times of the experiment. The present analysis reveals that this feature actually appears with time, while X1 deactivates in 4 ps. An interesting question is whether X1 and Y1 are excitedstate or ground-state species. From inspection of their SADS, it cannot be ruled out that they could display some stimulated emission and, hence, be excited-state species. On the other hand, it is clear that they do not exhibit the characteristic vibrational structure of stimulated emission at 660 nm (see OxyBP and Z1 in Figure 3). Note that if X1 were a ground-state species it would necessarily result from an unresolved, hence, subpicosecond precursor step. 3.1.2. TentatiVe Assignment of the Transient Species. It had previously been suggested that intermolecular electron transfer could be involved in the photophobic response of B. japonicum.12 In a former paper, we found a qualitative agreement between the specific transient absorption band of OBIP around 680 nm and the spectral signature of the OxyBP radical cation (OxyBP•+).20 The spectrum of OxyBP•+ was recorded by photooxidation of free OxyBP in ethanol in the presence of an electron acceptor, 1,4-benzoquinone (BZQ). Note that ethanol was chosen for this experiment in order to ensure that no groundstate complex between OxyBP and BZQ forms although, as any other solvent, it does not properly simulate the local environment of OxyBP within OBIP. We will see below how to take this difference into account. In the present work, it is now possible to make a more detailed comparison, with pure SADS. Figure 4 shows that the best match is obtained between OxyBP•+ and species Y1. We underline that this comparison is both more accurate and stringent than the approximate one done in our previous report.20 The present OxyBP•+ difference spectrum was recorded in ethanol where the steady-state absorption spectrum of OxyBP is blue-shifted by 13 nm as compared to the steadystate absorption spectrum of OBIP. This shift is particularly clear in Figure 4 around 550-560 nm where the first vibrational overtone of the lowest absorption transition is located, seen as

Oxyblepharismin-Binding Protein Photoprocesses

J. Phys. Chem. B, Vol. 111, No. 4, 2007 693

SCHEME 2: Kinetic Model for OxyBP in DMSOa

a Here, 100% of the X2 population decays to Y2, which decays to the triplet state Z2 with a yield of 25%.

a negative dip. It is striking that an almost identical shift is also seen around 670-680 nm, for the fingerprint peak of both OxyBP•+ and Y1 species. In other words, shifting one of the spectra by 13 nm yields a remarkable overlap of the two. It is nevertheless obvious that a full overlap is not achieved. The SADS of Y1 exhibits a positive transient absorption band between 480 and 550 nm while the difference absorption spectrum assigned to OxyBP•+ does not. This sizable difference might however be assigned to the spectral signature of the anion radical associated with the acceptor involved in the putative intermolecular electron transfer of OBIP. It had previously been suggested that such an acceptor could be a disulphide bridge (cystine) of the apoprotein.12,13 This suggestion can indeed be tentatively retained because the radical anion of a cystine has been reported to absorb between 350 and 550 nm at pH 7.31 As for the remaining transient absorption band that appears around 800 nm in the SADS of Y1, it may tentatively indicate the presence of a residual contribution of the initially excited state of rOBIP. Species Y1 would then be assigned to the OxyBP•+/ acceptor•- pair. Identifying Y1 with the OxyBP•+/acceptor•- pair implies an important consequence. It means that the OxyBP radical cation would be produced in the 4-ps regime and that it would have an observed precursor: X1. The nature of X1 is uncertain, but the likeness of the SADS of X1 and Y1 (Figure 2) points to similar species. It is important to recall that OxyBP•+ was produced by photooxidizing OxyBP in the presence of BZQ,20 and its difference absorption spectrum was recorded for a 10ns delay between the excitation and probe pulses. Consequently, one cannot exclude the possibility that the rapidly produced OxyBP•+ species could react on this long time scale. One can in particular imagine that a proton could be released by OxyBP•+, as was previously suggested.12,13 According to this hypothesis, species Y1 would in turn have to be identified with the deprotonated radical cation of OxyBP. In that case, species X1 could be associated with the simply oxidized form of OxyBP and reaction X1 f Y1 would be the conjectured deprotonation step of the radical cation. Considering these tentative attributions, we will discuss possible schemes for OBIP photoactivity in Blepharisma japonicum in section 3.3. 3.2. OxyBP in DMSO and Complexed with HSA. In this section, we briefly review the target analysis performed on previously reported20 difference absorption spectra of OxyBP, both in solution and complexed with human serum albumin (HSA). In the case of OxyBP dissolved in DMSO, the SVD-based procedure led to a sum of two exponentials, the lifetimes of which are 15 ps and 1 ns, and a step function. These values are nearly identical to the ones formerly determined20 by global analysis at 20 selected wavelengths (16 ps and 1.1 ns). We chose to describe those data with a simple three-state cascading model (Scheme 2). The initial excited state of OxyBP is represented by species X2 which decays in 15 ps toward state Y2. Y2 then partly returns to the ground state and partly produces a longlived species noted as Z2. The fraction R of the Y2 population

Figure 5. SADS of the X2, Y2, and Z2 species involved in the threestate cascading model (Scheme 2) describing OxyBP in DMSO.

that deactivates toward Z2 was found to be 0.25 ( 0.04. Figure 5 displays the SADS of X2, Y2, and Z2. The very slight differences betweeen the SADS of X2 and Y2 should be considered as significant and at the origin of the weak 15-ps component detected in the kinetics. We previously proposed6,20 that this component reflects an excited-state conformational relaxation of the molecule, a slight change of its nonplanar chiral32,33 geometry. As for state Z2, its difference spectrum is clearly assigned to the triplet state of OxyBP, which we recently published.20 Its characteristic shape is that of a broad T1Tn absorption band centered between 450 and 600 nm on which sharp bleaching structures are superimposed. The present analysis assigns to the triplet yield a new value, R ) 0.25, about twice as large as the one (0.12) formerly published by Ghetti for OxyBP in ethanol.34 In the preceding section, the excited-state behavior of nrOBIP was described by a single-exponential decay, while here three transient states are required to account for the photophysics of OxyBP in DMSO. In the OBIP kinetic traces, the presence of the large-amplitude picosecond decays assigned to rOBIP is likely to obscure the details of the nrOBIP dynamics on the short time scale, masking any free-OxyBP-like weak picosecond component. On the other hand, the production of a triplet state from the exited singlet Z1 state of nrOBIP could have been missed because of the S/N ratio of our experiment hardly allows the detection of more than three decay components. The 1.8-ns component of OBIP is in fact likely the monoexponential approximation of a more complex and longer decay, including the subnano- and nanosecond components previously observed by time-resolved fluorescence spectroscopy.19 Alternatively, the triplet yield of OxyBP within nrOBIP might also be reduced. We finally return to the question of the specificity of the interaction between OxyBP and its native protein partner. In a recent paper,20 we examined the transient absorption behavior of an artificial complex between OxyBP and HSA. The present analysis of these data by the SVD procedure yields a sum of two exponentials and a step function. The exponential lifetimes, 30 and 945 ps, are very close to the 29- and 900-ps values previously found by a global fit at 20 selected wavelengths. The simplest kinetic model suited to account for these results is actually identical to the one in Scheme 2 which confirms our previous qualitative conclusion that the OxyBP-HSA complex is much more similar to free OxyBP in solution than to OBIP.20 Such an element supports the idea that the OxyBP-protein interactions within OBIP bear at least a certain degree of specificity and that OBIP is a specialized macromolecular

694 J. Phys. Chem. B, Vol. 111, No. 4, 2007

Figure 6. SADS of the X3, Y3, and Z3 species involved in the threestate cascading model, similar to Scheme 2 for OxyBP, describing the OxyBP-HSA complex.

assembly. The three species involved are here noted X3, Y3, and Z3 and are tentatively assigned in the same way as those for OxyBP in DMSO. Figure 6 displays the SADS. The assignment of Z3 to the triplet state of the chromophore is however not so straightforward because its SADS displays noticeable differences with the difference absorption spectrum of the triplet state of free OxyBP in DMSO.20 A negative structure is in particular seen around 660 nm, in a region assigned to stimulated emission for the excited singlet state of OxyBP. This structure is not expected for the triplet state of OxyBP. The formation yield of Z3 was adjusted to R ) 0.65 ( 0.1. If Z3 were really assigned to the triplet state of OxyBP, this yield would be considerably larger than that of free OxyBP in DMSO (see above). Taking into account the fact that the fluorescence quantum yield of the OxyBP-HSA complex is twice as low as that of free OxyBP in DMSO,20 it can be deduced that the intersystem conversion rate would be increased by a factor of 4 in the HSA environment. Such an acceleration might be explained by a heavy atom effect, possibly due to the nearby presence of the sulfur atom of a cysteine or methionine residue.35 No other interpretation of the nature of Z3 is available to us at the moment. 3.3. Photoreactivity of OBIP in Blepharisma japonicum. In section 3.1, we have shown that the photoinduced processes in OBIP are well described by Scheme 1 and tentatively attributed the Y1 transient species observed for rOBIP either to the OxyBP•+ radical cation or to its deprotonated form. If this last hypothesis were true, one would need to admit that X1 results from an electron-transfer reaction occurring in the subpicosecond regime, within the time resolution of the present experimental setup. Such an ultrafast electron transfer would require the electron donor and acceptor to be situated at a very short distancesand probably with an optimized relative orientation. An example of this situation is the secondary photoinduced electron transfer in the photosynthetic reaction center of Rhodopseudomonas Viridis, reported to occur in 0.65 ps between the monomeric BA bacteriochlorophyl and the neighboring HA bacteriopheophytin.36,37 This geometrical constraint would imply that rOBIP is a rather rigid complex for which electron transfer has been optimized. In contrast, nrOBIP should be identified with the loose complex observed by Angelini by time-resolved fluorescence anisotropy, for which the excited chromophore is free to (slowly) rotate in all directions inside the protein pocket.6 Let us now focus our attention on the decay of species Y1 in 56 ps. Within the electron-transfer hypothesis, this process should correspond to the time it takes for Y1 to retrieve an

Plaza et al. electron. If Y1 were thought to be associated with the deprotonated OxyBP•+ radical cation, a proton should as well be retrieved during the 56-ps decay. In that special case, one can imagine that the recovered proton could be different from the one released by the chromophore during the primary steps. As in the case of bacteriorhodopsin, a neighboring protein residue could provide this hypothetical proton.38 This would allow one to propose that (reactive) OBIP is, like bacteriorhodopsin, a photoactivated proton pump, able to transfer protons across a membrane. The existence of such a pump could provide an interesting explanation to the translocation of protons across the membrane of pigment-containing granules, reported by Matsuoka.14,15 Let us recall that an intracellular pH drop appears to be at the origin of the phototransduction chain, possibly eliciting the depolarization of the cell membrane.39,40 Regardless of the above hypotheses on the nature of species X1 and Y1, it is quite striking that rOBIP undergoes such a fast photocycle, proposed here to be completed in a few hundred picoseconds. The expected function of a sensory photoreceptor is to produce a signaling state, the lifetime of which is long enough to trigger the transduction chain. For comparison, the signaling state of bovine rhodopsin (Meta II) has a lifetime of the order of 1 min.41 In a similar way, the signaling state (I2) of PYP (photoactive yellow protein, responsible for the negative phototaxis of Halorhodospira halophila) decays in a fraction of a second.42 One can nevertheless consider that a sensory photoreceptor protein is not necessarily bound to produce a signaling state for an external partner. A photoreceptor can also be an effector itself as in the case of PAC (photoactivated adenylyl cyclase, responsible for the step-up photophobic response of Euglena gracilis) which displays a photoinduced enzymatic activity.43,44 OBIP might fall into that category, but 56 ps is still a very short time to perform the desired biochemical reaction, regardless of its nature, and to restore the initial state of the system. Going back to the notion of proton translocation, it is interesting to note that deprotonation of retinal occurs in 50-100 µs in bacteriorhodopsin and its back-protonation is accomplished in 5-10 ms.45 Similarly, protonation of parahydroxycinnamic acid occurs in ca. 500 µs in PYP and its backdeprotonation happens in ca. 500 ms.42,46 As far as electron transfer is concerned, a long photocycle is also observed in the case of the photoactivation step of E. coli DNA photolyase. This reaction involves an initial fast (40-ps) electron transfer from a tryptophan residue to the excited flavin. In vitro, i.e., in the absence of external electron donors able to stabilize the charge-separated species, back electron transfer from the flavin to the proximal (4.2 Å) W382 residue is observed in the microsecond time scale.47 The time scale discrepancy between the above examples and the case of OBIP could be overcome suggesting that rOBIP undergoes a very fast initial photocycle of its chromophore that somehow triggers longer and spectroscopically dark steps, possibly localized on other domains of the protein. One may nevertheless consider an alternative interpretation. It should indeed not be excluded that the reactions we observed in the picosecond regime do not play any active role in the phototransduction chain of B. japonicum. If an electron transfer, accompanied or not by proton transfer, were involved in those steps, one could imagine that the ground-state recovery in 56 ps would just invert the initial transfers, the electron/proton acceptor partner now playing the role of donor. This would explain why both the contributions of the OxyBP radical cation and the corresponding radical anion simultaneously disappear during the decay of species Y1. Such a mechanism would of

Oxyblepharismin-Binding Protein Photoprocesses course not produce any long-lived photoproduct, nor would it induce any modification of the structure of the protein. Its function would be to rapidly consume photons, decreasing the excited-state concentration of OxyBP. It must be here recalled that OxyBP and BP are strong photosensitizers. They produce singlet oxygen with a high yield by energy transfer from their triplet state.48,49 It is even thought that this mechanism is a protection against predators and that the photophobic response is a way to escape from endogenous singlet oxygen production.50 Since B. japonicum converts to the blue OxyBP-containing form when it cannot escape from light, it would make sense that the fast photoprocess at work in rOBIP simply contributes to reducing the production of lethal singlet oxygen. This primary protection would be supplemented by OBIP efficiently encapsulating the chromophore and shielding it from molecular oxygen, as Checcucci et al. previously showed.49 4. Conclusion The aim of the present study was to quantify by target analysis our previously reported subpicosecond transient absorption spectra of OBIP, extract by this means the underlying photoinduced reaction mechanism, and thoroughly discuss the primary processes involved in this photoactive protein. The excited-state dynamics of OBIP is described by the sum of two contributions, corresponding to two classes of chromoprotein complex differing by the nature of the interaction between the chromophore and its protein environment. The nonreactive class (nrOBIP) is shown to be spectrally close to the free chromophore (OxyBP) in solution. The second class, called reactive OBIP (rOBIP), is attached to the specific features of OBIP, namely, the transient absorption band in the 680-nm region and the fast biexponential (4- and 56-ps) decay. The excited-state behavior of rOBIP is found to be well-described by a cascading scheme, 40% of the initial excited state (X1) giving rise to a transient state (Y1) that subsequently decays to the ground state. The OxyBP radical cation, which we reported in a previous paper,20 can be recognized in the Y1 transient state of rOBIP, in particular in the 680-nm region. Although the match is not perfect and the excited-state nature of Y1 cannot be entirely excluded, this element is an argument in favor of a previously published speculation, stating that an excited-state intermolecular electron transfer could be the primary step of the sensory transduction chain of B. japonicum.12 Different variants over this interpretation have been hypothesized, including proton transfer following electron transfer, but in all cases, a complete recovery of the initial ground state is achieved with a lifetime of 56 ps. Considering that the initially transferred electron and perhaps proton are being retrieved to the chromophore by different partners than the ones involved in the initial reactions, we have discussed that rOBIP acts as an electron or proton pump, which generates a signal or an electrochemical potential triggering the transduction chain. Alternatively considering that geminate recombination occurs, we have proposed a new biological function for rOBIP. It would thus merely be to absorb photons and convert them into heat. In other words, rOBIP would act as a solar screen, protecting the blue form of B. japonicum from excessive illumination. This hypothesis finds some support in the particularly fast overall photocycle of rOBIP, which does not seem a priori favorable to the building of a long-lived signaling state or to the production of secondary messengers. Acknowledgment. This work has been performed within the framework of a bilateral CNR-CNRS Exchange Project. We

J. Phys. Chem. B, Vol. 111, No. 4, 2007 695 also thank Prof. Dr. N.P. Ernsting (Institut fu¨r Chemie, Humboldt-Universita¨t zu Berlin) for kindly providing us enlightening tutorials on SVD-based analysis of transient absorption data. References and Notes (1) Kraml, M.; Marwan, W. Photochem. Photobiol. 1983, 37, 313. (2) Ghetti, F. Photoreception and photomovements in Blepharisma japonicum. In Biophysics of Photoreceptors and PhotomoVements in Microorganisms; Lenci, F., Colombetti, G., Ghetti, F., Ha¨der, D.-P., Song, P.-S., Eds.; NATO ASI Series A, Vol. 211; Plenum: New York, 1991; pp 257-266. (3) Giese, A. C. J. Cell. Comp. Physiol. 1946, 28, 119. (4) Matsuoka, T.; Sato, M.; Maeda, M.; Naoki, H.; Tanaka, T.; Kotsuki, H. Photochem. Photobiol. 1997, 65, 915. (5) Matsuoka, T.; Tokumori, D.; Kotsuki, H.; Ishida, M.; Matsushita, M.; Kimura, S.; Itoh, T.; Checcucci, G. Photochem. Photobiol. 2000, 72, 709. (6) Plaza, P.; Mahet, M.; Martin, M. M.; Angelini, N.; Malatesta, M.; Checcucci, G.; Lenci, F. Photochem. Photobiol. Sci. 2005, 4, 754. (7) Checcucci, G.; Damato, G.; Ghetti, F.; Lenci, F. Photochem. Photobiol. 1992, 57, 686. (8) Matsuoka, T.; Matsuoka, S.; Yamaoka, Y.; Kuriu, T.; Watanabe, Y.; Takayanagi, M.; Kato, Y.; Taneda, K. J. Protozool. 1992, 39, 498. (9) Scevoli, P.; Bisi, F.; Colombetti, G.; Ghetti, F.; Lenci, F.; Passarelli, V. J. Photochem. Photobiol. B: Biol. 1987, 1, 75. (10) Fabczak, H.; Fabczak, S.; Song, P.-S.; Checcucci, G.; Ghetti, F.; Lenci, F. J. Photochem. Photobiol. B: Biol. 1993, 21, 47. (11) Ghetti, F.; Checcucci, G.; Lenci, F. J. Photochem. Photobiol. B: Biol. 1992, 15, 185. (12) Angelini, N.; Quaranta, A.; Checcucci, G.; Song, P.-S.; Lenci, F. Photochem. Photobiol. 1998, 68, 864. (13) Sgarbossa, A.; Checcucci, G.; Lenci, F. Photochem. Photobiol. Sci. 2002, 1, 459. (14) Matsuoka, T.; Kotsuki, H. J. Exp. Zool. 2001, 289, 467. (15) Matsuoka, T.; Muraka, Y.; Furukohri, T.; Ishida, M.; Taneda, K. Photochem. Photobiol. 1992, 56, 399. (16) Walker, E. B.; Yoon, M.; Song, P.-S. Biochim. Biophys. Acta 1981, 634, 289. (17) Babcock, G. T.; Barry, B. A.; Debus, R. J.; Hoganson, C. W.; Atamian, M.; McIntosh, L.; Sithole, I.; Yocum, C. F. Biochemistry 1989, 28, 9557. (18) Falk, H. M. H. Angew. Chem., Int. Ed. 1999, 38, 3116. (19) Mahet, M.; Plaza, P.; Martin, M. M.; Checcucci, G.; Lenci, F. Fast excited-state reaction in the photoreceptor pigment-protein complex of the ciliate Blepharisma japonicum. In Femtochemistry and Femtobiology. Ultrafast EVents in Molecular Science; Martin, M. M., Hynes, J. T., Eds.; Elsevier Science: New York, 2004; pp 441-444. (20) Mahet, M.; Plaza, P.; Martin, M. M.; Checcucci, G.; Lenci, F. J. Photochem. Photobiol. A: Chem., published online Aug 1, http://dx.doi.org/ 10.1016/j.jphotochem.2006.06.030. (21) van Stokkum, I. H. M.; Larsen, D. S.; van Grondelle, R. Biochim. Biophys. Acta 2004, 1657, 82. (22) Senthil, V.; L., J. W.; G., C. A.; L., G. Biochim. Biophys. Acta 1992, 1115, 192. (23) Miskovsky, P.; Jancura, D.; Sanchez-Cortes, S.; Kocisova, E.; Chinsky, L. J. Am. Chem. Soc. 1998, 120, 6374. (24) Miskovsky, P.; Hritz, J.; Sanchez-Cortes, S.; Fabriciova, G.; Ulicny, J.; Chinsky, L. Photochem. Photobiol. 2001, 74, 172. (25) Falk, H.; Meyer, J. Monatsh. Chem. 1994, 125, 753. (26) Miskovsky, P. Curr. Drug Targets 2002, 3, 55. (27) Das, K.; Smirnov, A. V.; Wen, J.; Miskovsky, P.; Petrich, J. W. Photochem. Photobiol. 1999, 69, 633. (28) Henry, E. R.; Hofrichter, J. Methods Enzymol. 1992, 210, 129. (29) Penrose, R. Proc. Cambridge Philos. Soc. 1955, 51, 406. (30) Ernsting, N. P.; Kovalenko, S. A.; Senyushkina, T.; Saam, J.; Farztdinov, V. J. Phys. Chem. A 2001, 105, 3445. (31) Mezyk, S. P. Chem. Phys. Lett. 1995, 235, 89. (32) Pieroni, O.; Plaza, P.; Mahet, M.; Angelini, N.; Checcucci, G.; Malatesta, M.; Martin, M. M.; Lenci, F. Photochem. Photobiol. 2005, 81, 1343-1346. (33) Kida, A.; Takada, Y.; Kotsuki, H.; Tokumori, D.; Checcucci, G.; Matsuoka, T. Microbios 2001, 106, 189. (34) Ghetti, F.; Checcucci, G.; Lenci, F. J. Photochem. Photobiol. B: Biol. 1992, 13, 315. (35) Dro¨ssler, P.; Holzer, W.; Penzkofer, A.; Hegemann, P. Chem. Phys. 2003, 286, 409. (36) Zinth, W.; Artl, T.; Wachtveitl, J. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 1962. (37) Zinth, W.; Wachtveitl, J. Chem. Phys. Chem. 2005, 6, 871.

696 J. Phys. Chem. B, Vol. 111, No. 4, 2007 (38) Bondar, A. N.; Elstner, M.; Suhai, S.; Smith, J. C.; Fischer, S. Structure 2004, 12, 1281. (39) Fabczak, S.; Fabczak, H.; Song, P.-S. Photochem. Photobiol. 1993, 57, 872. (40) Kida, A.; Hase, M.; Tokumori, D.; Takada, Y.; Yasuda, M.; Kotsuki, H.; Checcucci, G.; Matsuoka, S. Trends Photochem. Photobiol. 2003, 10, 93. (41) Hofman, K. P. Late photoproducts and signalling states of bovine rhodopsin. In Molecular Mechanisms in Visual transduction; Stavenga, D. G., de Grip, W. J., Pugh, E. N., Eds.; Elsevier/North Holland: New York, 2000; pp 91-142. (42) Hellingwerf, K. J.; Hendriks, J.; Gensch, T. J. Phys. Chem. A 2003, 107, 1082. (43) Iseki, M.; Matsunaga, S.; Murakami, A.; Ohno, K.; Shiga, K.; Yoshida, K.; Sugai, M.; Takahashi, T.; Terumitsu, H.; Watanabe, M. Nature 2002, 415, 1047.

Plaza et al. (44) Yoshikawa, S.; Suzuki, T.; Watanabe, M.; Iseki, M. Photochem. Photobiol. Sci. 2005, 4, 727. (45) Mathew, M. K. Curr. Sci. 2002, 83, 799. (46) Changenet-Barret, P.; Espagne, A.; Plaza, P.; Hellingwerf, K. J.; Martin, M. M. New J. Chem. 2005, 29, 527. (47) Byrdin, M.; Sartor, V.; Eker, A. P. M.; Vos, M. H.; Aubert, C.; Brettel, K.; Mathis, P. Biochim. Biophys. Acta 2004, 1655, 64. (48) Checcucci, G.; Lenci, F.; Ghetti, F.; Song, P.-S. J. Photochem. Photobiol. B: Biol. 1991, 11, 49. (49) Checcucci, G.; Takada, Y.; Matsuoka, T. Mem. Fac. Sci., Kochi UniV. Ser. D, Biol. 2001, 22, 39. (50) Terazima, M. N.; Iio, H.; Harumoto, T. Photochem. Photobiol. 1999, 69, 47.