Portal de Periódicos da CAPES

Lutein Can Act as a Switchable Charge Transfer Quencher in the CP26 Light-harvesting Complex

2008; Elsevier BV; Volume: 284; Issue: 5 Linguagem: Inglês

10.1074/jbc.m807192200

1083-351X

Thomas J. Avenson, Tae Kyu Ahn, Krishna Niyogi, Matteo Ballottari, Roberto Bassi, Graham R. Fleming,

Plant and animal studies

Energy-dependent quenching of excitons in photosystem II of plants, or qE, has been positively correlated with the transient production of carotenoid radical cation species. Zeaxanthin was shown to be the donor species in the CP29 antenna complex. We report transient absorbance analyses of CP24 and CP26 complexes that bind lutein and zeaxanthin in the L1 and L2 domains, respectively. For CP24 complexes, the transient absorbance difference profiles give a reconstructed transient absorbance spectrum with a single peak centered at ∼980 nm, consistent with zeaxanthin radical cation formation. In contrast, CP26 gives constants for the decay components probed at 940 and 980 nm of 144 and 194 ps, a transient absorbance spectrum that has a main peak at 980 nm, and a substantial shoulder at 940 nm. This suggests the presence of two charge transfer quenching sites in CP26 involving zeaxanthin radical cation and lutein radical cation species. We also show that lutein radical cation formation in CP26 is dependent on binding of zeaxanthin to the L2 domain, implying that zeaxanthin acts as an allosteric effector of charge transfer quenching involving lutein in the L1 domain. Energy-dependent quenching of excitons in photosystem II of plants, or qE, has been positively correlated with the transient production of carotenoid radical cation species. Zeaxanthin was shown to be the donor species in the CP29 antenna complex. We report transient absorbance analyses of CP24 and CP26 complexes that bind lutein and zeaxanthin in the L1 and L2 domains, respectively. For CP24 complexes, the transient absorbance difference profiles give a reconstructed transient absorbance spectrum with a single peak centered at ∼980 nm, consistent with zeaxanthin radical cation formation. In contrast, CP26 gives constants for the decay components probed at 940 and 980 nm of 144 and 194 ps, a transient absorbance spectrum that has a main peak at 980 nm, and a substantial shoulder at 940 nm. This suggests the presence of two charge transfer quenching sites in CP26 involving zeaxanthin radical cation and lutein radical cation species. We also show that lutein radical cation formation in CP26 is dependent on binding of zeaxanthin to the L2 domain, implying that zeaxanthin acts as an allosteric effector of charge transfer quenching involving lutein in the L1 domain. Regulation of light capture during photosynthesis occurs primarily within the antenna of photosystem II, the peripheral portion of which is comprised of trimeric light-harvesting complex (LHC) II 4The abbreviations used are: LHC, light-harvesting complex; qE, energy-dependent quenching; NPQ, non-photochemical quenching; CT, charge transfer; Chl, chlorophyll; Lut, lutein; Z, zeaxanthin; V, violaxanthin; TA, transient absorbance; NIR, near infrared region; Car, carotenoid; HPLC, high pressure liquid chromatography.4The abbreviations used are: LHC, light-harvesting complex; qE, energy-dependent quenching; NPQ, non-photochemical quenching; CT, charge transfer; Chl, chlorophyll; Lut, lutein; Z, zeaxanthin; V, violaxanthin; TA, transient absorbance; NIR, near infrared region; Car, carotenoid; HPLC, high pressure liquid chromatography. (1Kuhlbrandt W. Wang D.N. Fujiyoshi Y. Nature.. 1994; 367: 614-621Google Scholar) and the monomeric minor LHCs CP24, CP26, and CP29 (2Andersson J. Walters R.G. Horton P. Jansson S. Plant Cell.. 2001; 13: 1193-1204Google Scholar). Regulation of light capture is critical for plant fitness (3Kulheim C. Agren J. Jansson S. Science.. 2002; 297: 91-93Google Scholar) and is achieved predominantly by a process termed energy-dependent quenching, or qE (4Kramer D.M. Avenson T.J. Edwards G.E. Trends Plant Sci... 2004; 9: 349-357Google Scholar), one of a composite of processes involved in the non-photochemical quenching (NPQ) of excess absorbed light energy (5Muller P. Li X.P. Niyogi K.K. Plant Physiol... 2001; 125: 1558-1566Google Scholar, 6Horton P. Ruban A.V. Walters R.G. Annu. Rev. Plant Physiol. Plant Mol. Biol... 1996; 47: 655-684Google Scholar, 7Demmig-Adams B. Gilmore A.M. Adams W.W. FASEB J.. 1996; 10: 403-412Google Scholar). Several characteristics distinguish qE from the other components of NPQ. First, qE is reversible on the seconds-to-minutes time scale, a feat that is thought to reflect rapid changes in the thylakoid lumen pH, which transmit information to the antenna where the molecular mechanism of qE is modulated. Secondly, the npq1 and npq4 mutant strains of Arabidopsis thaliana, which lack the capacity for generating zeaxanthin (Z) (8Niyogi K.K. Grossman A.R. Björkman O. Plant Cell.. 1998; 10: 1121-1134Google Scholar) and the PsbS protein (9Li X.P. Björkman O. Shih C. Grossman A.R. Rosenquist M. Jansson S. Niyogi K.K. Nature.. 2000; 403: 391-395Google Scholar), respectively, exhibit very little qE, consistent with Z and PsbS being necessary for qE.The peripheral antenna is generally thought to be the location of the molecular mechanism of qE, although within precisely which of the LHCs, as well as by what molecular mechanism(s), are issues currently under intense investigation (10Holt N.E. Fleming G.R. Niyogi K.K. Biochemistry.. 2004; 43: 8281-8289Google Scholar, 11Horton P. Ruban A. J. Exp. Bot... 2005; 56: 365-373Google Scholar, 12Pascal A.A. Liu Z. Broess K. van Oort B. van Amerongen H. Wang C. Horton P. Robert B. Chang W. Ruban A. Nature.. 2005; 436: 134-137Google Scholar, 13Ruban A.V. Berera R. Ilioaia C. van Stokkum I.H. Kennis J.T. Pascal A.A. van Amerongen H. Robert B. Horton P. van Grondelle R. Nature.. 2007; 450: 575-578Google Scholar). Assigning where and by what mechanism(s) qE occurs is hindered by the fact that qE is, by definition, a phenomenon requiring an intact system, complicating purely deconstructive approaches for studying this mechanism. Nonetheless, several recent studies have combined various approaches to correlate the phenomenology of qE with various components and molecular mechanisms. For example, it was recently shown that excitation energy transfer from singlet-excited chlorophyll (Chl) to the S1 state of lutein (Lut) occurs both within isolated LHCII trimeric complexes (13Ruban A.V. Berera R. Ilioaia C. van Stokkum I.H. Kennis J.T. Pascal A.A. van Amerongen H. Robert B. Horton P. van Grondelle R. Nature.. 2007; 450: 575-578Google Scholar), previously hypothesized to be a site for qE (14Standfuss J. Terwisscha van Scheltinga A.C. Lamborghini M. Kuhlbrandt W. EMBO J.. 2005; 24: 919-928Google Scholar), and in vivo in a qE-dependent manner (13Ruban A.V. Berera R. Ilioaia C. van Stokkum I.H. Kennis J.T. Pascal A.A. van Amerongen H. Robert B. Horton P. van Grondelle R. Nature.. 2007; 450: 575-578Google Scholar). Evidence for charge transfer (CT) quenching of Chl excited states, a mechanism previously predicted from quantum chemical calculations (15Dreuw A. Fleming G.R. Head-Gordon M. Phys. Chem. Chem. Phys... 2003; 5: 3247-3256Google Scholar, 16Dreuw A. Fleming G.R. Head-Gordon M. Biochem. Soc. Trans... 2005; 33: 858-862Google Scholar), within a Chl-Z heterodimer ((Chl-Z)) complex has been positively correlated with all of the phenomenological aspects of qE using isolated thylakoids (17Avenson T.J. Ahn T.K. Zigmantas D. Niyogi K.K. Li Z. Ballottari M. Bassi R. Fleming G.R. J. Biol. Chem... 2008; 283: 3550-3558Google Scholar, 18Holt N.E. Zigmantas D. Valkunas L. Li X.P. Niyogi K.K. Fleming G.R. Science.. 2005; 307: 433-436Google Scholar). Evidence for CT quenching, e.g. transient Z radical cation (Z•+) formation (17Avenson T.J. Ahn T.K. Zigmantas D. Niyogi K.K. Li Z. Ballottari M. Bassi R. Fleming G.R. J. Biol. Chem... 2008; 283: 3550-3558Google Scholar, 18Holt N.E. Zigmantas D. Valkunas L. Li X.P. Niyogi K.K. Fleming G.R. Science.. 2005; 307: 433-436Google Scholar), has been demonstrated in a composite mixture of CP24, CP26, and CP29 complexes (17Avenson T.J. Ahn T.K. Zigmantas D. Niyogi K.K. Li Z. Ballottari M. Bassi R. Fleming G.R. J. Biol. Chem... 2008; 283: 3550-3558Google Scholar), and recently, in isolated CP29 complexes (19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar). Estimates suggest that CT quenching within all three minor complexes can account for a significant fraction of qE in isolated thylakoids (17Avenson T.J. Ahn T.K. Zigmantas D. Niyogi K.K. Li Z. Ballottari M. Bassi R. Fleming G.R. J. Biol. Chem... 2008; 283: 3550-3558Google Scholar), although it should be emphasized that simultaneous operation of multiple mechanisms throughout the antenna during qE is not excluded.The molecular architecture of the CT site in CP29 has recently been elucidated (19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar). Based on homology with the LHCII complex, eight Chl and two carotenoid (Car) binding sites have been assigned to CP29, all of which reside within two protein domains referred to as L1 and L2 (20Bassi R. Croce R. Cugini D. Sandona D. Proc. Natl. Acad. Sci. U. S. A... 1999; 96: 10056-10061Google Scholar). Lut and the xanthophyll cycle carotenoids (Z and violaxanthin, or V) preferentially bind to the L1 and L2 sites, respectively (21Formaggio E. Cinque G. Bassi R. J. Mol. Biol... 2001; 314: 1157-1166Google Scholar). Chls a that bind to sites A5 and B5 (corresponding to Chl 5 and Chl 12, respectively, in LHCII (14Standfuss J. Terwisscha van Scheltinga A.C. Lamborghini M. Kuhlbrandt W. EMBO J.. 2005; 24: 919-928Google Scholar)) are excitonically coupled (20Bassi R. Croce R. Cugini D. Sandona D. Proc. Natl. Acad. Sci. U. S. A... 1999; 96: 10056-10061Google Scholar), their interaction has been shown to be necessary for regulation of fluorescence lifetime (22Ihalainen J.A. Croce R. Morosinotto T. van Stokkum I.H. Bassi R. Dekker J.P. van Grondelle R. J. Phys. Chem. B... 2005; 109: 21150-21158Google Scholar, 23Morosinotto T. Breton J. Bassi R. Croce R. J. Biol. Chem... 2003; 278: 49223-49229Google Scholar) and Chl triplet quenching in the homologous protein Lhca4 (24Carbonera D. Agostini G. Morosinotto T. Bassi R. Biochemistry.. 2005; 44: 8337-8346Google Scholar), and they have been shown to be involved in CT quenching in a (Chl-Z) complex within the L2 domain (19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar).The CP26 complex provides an opportunity to assess whether other Car species can be involved in CT quenching. Lut is bound at the L1 site, which also holds a pair of Chl binding sites (A2 and B2 corresponding to Chl 2 and Chl 7, respectively in LHCII (14Standfuss J. Terwisscha van Scheltinga A.C. Lamborghini M. Kuhlbrandt W. EMBO J.. 2005; 24: 919-928Google Scholar)), both close to the Car and to each other (25Mozzo M. Passarini F. Bassi R. van Amerongen H. Croce R. Biochim. Biophys. Acta.. 2008; 1777: 1263-1267Google Scholar), the features found for the L2 site and Chls A5 and B5 in CP29 (19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar).In this report, we describe transient absorbance (TA) analyses of CP24 and CP26 complexes with various Cars bound to sites L1 and L2 by excitation of the complexes at the Chl Qy transition and probing for transient species within the near infrared region (NIR), as in Refs. 17Avenson T.J. Ahn T.K. Zigmantas D. Niyogi K.K. Li Z. Ballottari M. Bassi R. Fleming G.R. J. Biol. Chem... 2008; 283: 3550-3558Google Scholar, 18Holt N.E. Zigmantas D. Valkunas L. Li X.P. Niyogi K.K. Fleming G.R. Science.. 2005; 307: 433-436Google Scholar, 19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar. The results suggest that although a single CT quenching site involving a Z•+ species operates within CP24, presumably within the L2 domain, two CT sites exist within CP26, one of which involves a Z•+ species within the L2 domain and the other a Lut radical cation (Lut•+) within the L1 domain. We discuss these results in the context of the different CT site architectures of the CP29 and CP26 complexes.MATERIALS AND METHODSIsolation of Antenna LHCs with Specific Xanthophylls—The genes for CP24 and CP26 polypeptides from A. thaliana were expressed in Escherichia coli, and the apoproteins were isolated followed by in vitro reconstitution with Chls (a and b), neoxanthin, Lut, and either V or Z. Pigments were extracted from the isolated antenna complexes with 80% acetone and then separated and quantified by HPLC as described in Ref. 26Gilmore A.M. Yamamoto H.Y. Plant Physiol... 1991; 96: 635-643Google Scholar and by fitting analysis of the spectrum of the acetone extract with the spectra of individual pigments as described in Ref. 27Croce R. Canino G. Ros F. Bassi R. Biochemistry.. 2002; 41: 7334-7343Google Scholar.Transient Absorbance Setup—A femtosecond TA laser system that was recently described in detail was used to perform NIR TA analyses (17Avenson T.J. Ahn T.K. Zigmantas D. Niyogi K.K. Li Z. Ballottari M. Bassi R. Fleming G.R. J. Biol. Chem... 2008; 283: 3550-3558Google Scholar). Briefly, excitation pulses were centered at 650 nm, and the probe region used was 880–1080 nm. CP24 and CP26 complexes were resuspended in buffer solution (5 mm HEPES and 0.06% α-dodecylmaltocide at pH 7.6) to an OD of ∼0.3/mm (all samples were adjusted to the same OD at 650 nm) for TA analyses. A sample cell with a path length of 1 mm was chilled by a circulating water bath (VWR Scientific 1160, PolyScientific, Niles, IL), which was set at 7 °C during the data acquisition to prevent sample degradation.RESULTSCarotenoid Compositions of Isolated CP24 and CP26 Apoproteins—Isolated recombinant apoproteins of CP24 and CP26 overexpressed in E. coli were reconstituted in vitro with Chls (a and b), neoxanthin, Lut, and either V or Z, or in some instances, only with the individual Cars (below). Table 1 shows that, as determined by HPLC analyses, the CP24 complexes reconstituted in the presence of V (referred to herein as CP24V) contained 0.6 V and 1.6 Lut molecules per 10 Chls, respectively. In contrast, the CP24Z complexes consisted of 1.4 Z and 0.8 Lut molecules, respectively. The CP26V complexes were comprised of 0.2 V and 1.6 Lut molecules, respectively, whereas the CP26Z complexes consisted of 1.1 Z and 1.1 Lut molecules, respectively. The minor complexes preferentially bind Lut and Z (or V) in the L1 and L2 domains, respectively (27Croce R. Canino G. Ros F. Bassi R. Biochemistry.. 2002; 41: 7334-7343Google Scholar).TABLE 1Pigment compositions of reconstituted CP24 and CP26 complexesSample# of ChlsChl a/Chl bChl/CarNeoViolaLutZea# of CarCP26V92.23.50.80.21.60.02.6CP26Z92.04.20.00.01.11.12.1CP24V101.54.40.00.61.60.02.2CP24Z101.64.70.00.00.81.42.1 Open table in a new tab Variable Car•+ Formation in CP24 and CP26 Complexes—The Car radical cation (Car•+) species exhibits strong absorption in the NIR (28Galinato M.G. Niedzwiedzki D. Deal C. Birge R.R. Frank H.A. Photosynth. Res... 2007; 94: 67-78Google Scholar). Shown in Fig. 1 are TA difference spectra for CP24 (panel A) and CP26 (panel B) that were obtained, essentially as described in Refs. 17Avenson T.J. Ahn T.K. Zigmantas D. Niyogi K.K. Li Z. Ballottari M. Bassi R. Fleming G.R. J. Biol. Chem... 2008; 283: 3550-3558Google Scholar and 19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar, from a series of NIR TA kinetic traces by excitation of the CP24Z/CP24V and CP26Z/CP26V complexes at the Chl Qy transition and probing for transient Car•+ species from 880 to 1080 nm. The CP24 spectrum is characterized by broad absorption and a central peak at ∼970–980 nm, very similar to the TA spectrum obtained for CP29 and in which the 980 nm peak was assigned to transient Z•+ formation during CT quenching (17Avenson T.J. Ahn T.K. Zigmantas D. Niyogi K.K. Li Z. Ballottari M. Bassi R. Fleming G.R. J. Biol. Chem... 2008; 283: 3550-3558Google Scholar, 19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar, 29Amarie S. Standfuss J. Barros T. Kulbrandt W. Dreuw A. Wachtveitl J. J. Phys. Chem. B... 2007; 111: 3481-3487Google Scholar). In contrast to the single peak observed in the NIR TA spectra for CP24 and CP29 (19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar), the spectrum for CP26 exhibits, in addition to a peak at 980 nm, a shoulder at ∼950 nm. Although these combined results are consistent with the occurrence of CT quenching involving a Z•+ species in the L2 domain in all three minor complexes, as has been previously suggested (17Avenson T.J. Ahn T.K. Zigmantas D. Niyogi K.K. Li Z. Ballottari M. Bassi R. Fleming G.R. J. Biol. Chem... 2008; 283: 3550-3558Google Scholar, 19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar), the two peaks observed in the CP26 TA spectrum are consistent with the transient formation of multiple Car•+ species, possibly indicative of an auxiliary CT site. Recall that the L1 site of CP26Z is occupied by Lut, hinting that the alternative CT site may involve a Lut•+, previously shown to absorb at 940–950 nm (28Galinato M.G. Niedzwiedzki D. Deal C. Birge R.R. Frank H.A. Photosynth. Res... 2007; 94: 67-78Google Scholar). The area under the ∼940 nm band is ∼28% of the total absorbance, suggesting a somewhat lower yield of Lut cation, because Lut and Z bind to CP26 in a 1:1 ratio (Table 1), whereas the extinction coefficient of Lut•+ is 70% that of Z•+ (30Han R.-M. Tain Y.-X. Wu Y.-S. Wang P. Ai X.-C. Zhang J.-P. Skibsted L.H. Photochem. Photobiol. Sci... 2006; 82: 538-546Google Scholar, 31Mortensen A. Skibsted L.H. J. Agric. Food Chem... 1997; 45: 2970-2977Google Scholar).Transient Z•+ Formation in CP24 and CP26—CT quenching dynamics can be inferred from NIR TA kinetic analyses (17Avenson T.J. Ahn T.K. Zigmantas D. Niyogi K.K. Li Z. Ballottari M. Bassi R. Fleming G.R. J. Biol. Chem... 2008; 283: 3550-3558Google Scholar, 18Holt N.E. Zigmantas D. Valkunas L. Li X.P. Niyogi K.K. Fleming G.R. Science.. 2005; 307: 433-436Google Scholar, 19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar). Fig. 2A shows that the 980 nm TA kinetic profile for the CP24V sample (black trace) is characterized solely by decay components, essentially as was observed in CP29 bound by V (19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar) and attributed to the dynamics of Chl excited state absorbance (17Avenson T.J. Ahn T.K. Zigmantas D. Niyogi K.K. Li Z. Ballottari M. Bassi R. Fleming G.R. J. Biol. Chem... 2008; 283: 3550-3558Google Scholar, 18Holt N.E. Zigmantas D. Valkunas L. Li X.P. Niyogi K.K. Fleming G.R. Science.. 2005; 307: 433-436Google Scholar, 19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar). In contrast, the 980 nm TA kinetic profile for CP24Z complexes (red trace) exhibits multiexponential rise components followed by biphasic decay. The TA difference profile (blue trace), obtained by subtracting the 980 nm TA kinetics of the V-complexes from those of the Z counterparts, shows biphasic rise components, similar to those observed and kinetically modeled for CP29 (32Cheng Y.-C. Ahn T.K. Avenson T.J. Zigmantas D. Niyogi K.K. Ballottari M. Bassi R. Fleming G.R. J. Phys. Chem. B... 2008; 112: 13418-13423Google Scholar). The faster and slower rise components displayed time constants of <500 fs and∼5 ps, respectively. The kinetics of the sub-ps rise are attributable to either very fast intracomplex energy transfer to the CT quenching site or to excitation of the CT site directly, as discussed in Ref. 32Cheng Y.-C. Ahn T.K. Avenson T.J. Zigmantas D. Niyogi K.K. Ballottari M. Bassi R. Fleming G.R. J. Phys. Chem. B... 2008; 112: 13418-13423Google Scholar. The slower ∼5 ps rise component can be assigned to the dynamics of intracomplex energy transfer to the CT trap site (32Cheng Y.-C. Ahn T.K. Avenson T.J. Zigmantas D. Niyogi K.K. Ballottari M. Bassi R. Fleming G.R. J. Phys. Chem. B... 2008; 112: 13418-13423Google Scholar). The 980 nm signal in CP24 decays as a single exponential with a time constant of 103 ps, when compared with the 238-ps decay seen in CP29 at this wavelength (19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar). These results imply that the dynamics of energy transfer to the CT site are insensitive to the intrinsic variability of the L2 domains of CP24 and CP29, whereas the charge recombination of the respective charge-separated states is sensitive to the protein environment. The multiexponential rise components (<500 fs and 5 ps, respectively) of the 980 nm TA difference profile for CP26Z (Fig. 2B) are similar to those observed for both CP24Z and CP29Z (19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar), whereas the TA signal decays with a time constant of 194 ps, intermediate between the CP24Z and CP29Z values.FIGURE 2NIR TA kinetic profiles for CP24 and CP26 complexes probed at 980 nm. NIR TA profiles were obtained using isolated, monomeric CP24 (A) and CP26 (B) complexes by excitation at 650 nm and probing at 980 nm. The red and black traces represent TA kinetic profiles for the corresponding complexes bound by Z and V, respectively, and represent an average of more than 10 kinetic sweeps. Difference kinetic traces (blue) correspond to subtraction of the V-kinetic profiles from the Z-kinetics. a.u., arbitrary units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)If the Car•+ band at 940 nm is formed independently of the Z•+ 980 nm band, we expect different kinetics in the two spectral regions. Fig. 3A shows that the time constants of the biexponential rise components of the 940 nm TA difference profile for CP24 were <500 fs and 5 ps, respectively, and that the signal decays with a time constant of 108 ps. These CP24 TA dynamics are, within the noise level, the same as those observed at 980 nm (Fig. 2A), implying that the same Car•+ (i.e. Z•+) species is probed at the two wavelengths. Because all CP24 complexes bind two carotenoids, it is possible that some of the Z•+ signal comes from CP24 complexes containing Z in both L1 and L2 sites, although the kinetics would have to be identical in the two sites, which seems unlikely. However, the dominant (70% under this assumption) species is 1Lut/1Z, and we do not see a Lut•+ signal in this sample. The biphasic rise components of the 940 nm TA difference profile for CP26 are <500 fs and 5 ps (Fig. 3B), respectively, as was observed at 980 nm; however, the 940 nm signal decayed with a time constant of 144 ps, in contrast to 194 ps at 980 nm (Fig. 2B), supporting the idea of independent CT quenching sites in CP26 with Z in the L2 site and Lut in the L1 site.FIGURE 3940 nm TA difference profiles for isolated CP24 (A) and CP26 (B) complexes. NIR TA difference profiles were obtained as in Fig. 2 except that the probe wavelength was 940 nm. a.u., arbitrary units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Alternatively, the differential decay dynamics observed for CP26 at 940 and 980 nm, as well as the two-peak TA spectrum, could reflect protein environmentally induced variability in Z•+ formation within distinct populations of complexes that bind Z in both the L1 and the L2 domains. Fig. 4A shows NIR TA spectra that were obtained for CP26 complexes reconstituted solely with a single type of Car, i.e. V, Z, or Lut in both sites L1 and L2 (27Croce R. Canino G. Ros F. Bassi R. Biochemistry.. 2002; 41: 7334-7343Google Scholar). The pigment compositions of the complex are given in Table 2. The TA spectrum for CP26 reconstituted with only V does not exhibit absorption above the baseline throughout the region where Car•+ species typically absorb, confirming that V does not participate in CT quenching when bound to either L1 or L2 domains of CP26. Similarly, the TA spectrum for CP26 reconstituted with only Lut also shows no evidence for Car•+ formation, implying that CT quenching involving a Lut•+ is not supported when Lut occupies the L1 and L2 domains simultaneously. Occupancy of L2 by V, which competes for binding within this domain, apparently does not support a conformation of the protein that facilitates Lut•+ formation in the L1 site, as suggested by the lack of evidence for Car•+ formation in the spectrum for CP26 reconstituted with both V and Lut (data not shown). In contrast, Car•+ formation is clearly evident in the spectrum for CP26 reconstituted solely with Z, where broad absorption characterized by a single peak centered at ∼980 nm is observed. Furthermore, Fig. 4B shows that the difference spectrum, obtained by subtracting the spectrum of the complexes that bind V only from that of the complexes that were reconstituted solely with Z, exhibits a single peak at ∼980 nm, consistent with previously reported Z•+ absorption characteristics during CT quenching (17Avenson T.J. Ahn T.K. Zigmantas D. Niyogi K.K. Li Z. Ballottari M. Bassi R. Fleming G.R. J. Biol. Chem... 2008; 283: 3550-3558Google Scholar, 18Holt N.E. Zigmantas D. Valkunas L. Li X.P. Niyogi K.K. Fleming G.R. Science.. 2005; 307: 433-436Google Scholar, 19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar). These results imply either that Z•+ formation occurs in both sites L1 and L2 and the respective Z•+ species possess similar absorption maxima or that Z•+ formation occurs only in the L2 site (i.e. CT quenching involving a Z•+ does not occur in the L1 site when bound by Z).FIGURE 4Transient spectra for CP26 complexes reconstituted with single carotenoid species. A range of NIR TA kinetic profiles (i.e. obtained by probing from 880 to 1080 nm) was generated in isolated CP26 complexes that were reconstituted solely with V, Z, or Lut. A, spectra for complexes that bind Z (circles), V (triangles), and Lut (squares) were obtained as in Fig. 1. The V spectrum was multiplied by 1.5 for comparison. B, difference spectra for Z-complexes minus V-complexes (squares) and L-complexes minus V-complexes (circles). Error bars represent the S.E. of five time points.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2Pigment compositions of reconstituted CP26 complexes with single xanthophyll speciesSample# of ChlsChl a/Chl bNeoViolaLutZea# of CarCP26-Lut292.70.00.01.80.01.8CP26-V292.50.02.00.00.02.0CP26-Z292.70.00.00.01.51.5 Open table in a new tab Overall these results imply that the 940 nm band in CP26Z arises from Lut•+, not Z•+ in the L1 site. Binding of Z to the L2 site has been shown to produce a conformational change in CP26 (33Dall'Osto L. Caffarri S. Bassi R. Plant Cell.. 2005; 17: 1217-1232Google Scholar).DISCUSSIONZ Is an Allosteric Modulator of CT Quenching within a (Chl-Lut) in CP26—Z has been proposed to have multiple roles in dissipating excess absorbed light energy (1Kuhlbrandt W. Wang D.N. Fujiyoshi Y. Nature.. 1994; 367: 614-621Google Scholar, 11Horton P. Ruban A. J. Exp. Bot... 2005; 56: 365-373Google Scholar, 12Pascal A.A. Liu Z. Broess K. van Oort B. van Amerongen H. Wang C. Horton P. Robert B. Chang W. Ruban A. Nature.. 2005; 436: 134-137Google Scholar, 17Avenson T.J. Ahn T.K. Zigmantas D. Niyogi K.K. Li Z. Ballottari M. Bassi R. Fleming G.R. J. Biol. Chem... 2008; 283: 3550-3558Google Scholar, 18Holt N.E. Zigmantas D. Valkunas L. Li X.P. Niyogi K.K. Fleming G.R. Science.. 2005; 307: 433-436Google Scholar, 19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar, 34Ruban A.V. Solovieva S. Lee P.J. Ilioaia C. Wentworth M. Ganeteg U. Klimmek F. Chow W.S. Anderson J.M. Jansson S. Horton P. J. Biol. Chem... 2006; 281: 14981-14990Google Scholar). On the one hand, Z has been suggested to be directly involved in the quenching of Chl excited states during qE via CT quenching (18Holt N.E. Zigmantas D. Valkunas L. Li X.P. Niyogi K.K. Fleming G.R. Science.. 2005; 307: 433-436Google Scholar), evidence for which, e.g. transient Z•+ formation, was obtained in a composite mixture of all three minor complexes (17Avenson T.J. Ahn T.K. Zigmantas D. Niyogi K.K. Li Z. Ballottari M. Bassi R. Fleming G.R. J. Biol. Chem... 2008; 283: 3550-3558Google Scholar) and more recently in isolated CP29 (19Ahn T.K. Avenson T.J. Ballottari M. Cheng Y.C. Niyogi K.K. Bassi R. Fleming G.R. Science.. 2008; 320: 794-797Google Scholar, 32Cheng Y.-C. Ahn T.K. Avenson T.J. Zigmantas D. Niyogi K.K. Ballottari M. Bassi R. Fleming G.R. J. Phys. Chem. B... 2008; 112: 13418-13423Google Scholar) and in isolated CP24 and CP26 complexes (this work). It has also been suggested that transfer of energy from singlet-excited Chl to the S1 state of Z may occur, thereby allowing the direct quenching of Chl excites states (35Demmi