In situ formation of photoactive B-ring reduced chlorophyll isomer in photosynthetic protein LH2

Natural chlorophylls have a D-ring reduced chlorin π-system; however, no naturally occurring photosynthetically active B-ring reduced chlorins have been reported. Here we report a B-ring reduced chlorin, 17,18-didehydro-bacteriochlorophyll (BChl) a, produced by in situ oxidation of B800 bacteriochlorophyll (BChl) a in a light-harvesting protein LH2 from a purple photosynthetic bacterium Phaeospirillum molischianum. The regioselective oxidation of the B-ring of B800 BChl a is rationalized by its molecular orientation in the protein matrix. The formation of 17,18-didehydro-BChl a produced no change in the local structures and circular arrangement of the LH2 protein. The B-ring reduced 17,18-didehydro-BChl a functions as an energy donor in the LH2 protein. The photoactive B-ring reduced Chl isomer in LH2 will be helpful for understanding the photofunction and evolution of photosynthetic cyclic tetrapyrrole pigments.

compares the electronic absorption spectrum of molischianum-LH2 after incubation with DDQ (denoted as oxidized molischianum-LH2) with that of native LH2. A Q y absorption band at 799 nm of B800 BChl a was absent from the oxidized molischianum-LH2, and a new absorption band appeared at 700 nm. We attribute this feature to the Q y band of the oxidized pigment, produced from B800 BChl a. Difference spectrum of the oxidized/ native molischianum-LH2 revealed a Soret band for the oxidized pigment at 449 nm (Fig. S2). In the oxidation of B800 BChl a, the peak position and the bandwidth of the Q y band of B850 BChl a remained unchanged. The absorption spectrum of oxidized molischianum-LH2 barely changed by incubation with sodium ascorbate (100 mM), indicating the oxidation of B800 BChl a is irreversible (Fig. S3).
Spectral changes of oxidized molischianum-LH2 at 40 °C are compared with those of native LH2 to check the stability of oxidized LH2. Both the LH2 samples exhibited no change in their spectra (Fig. S4). These results  www.nature.com/scientificreports/ suggest that oxidized molischianum-LH2 is stable in a similar level to native LH2. The stability of oxidized molischianum-LH2 is also confirmed by no degradation of oxidized molischianum-LH2 during its purification and various measurements. The Q y peak position of the new pigment in oxidized molischianum-LH2 shifted to a longer wavelength than that of D-ring reduced 3-acetyl-Chl (AcChl) a (Fig. 1C) in the B800 site of molischianum-LH2 at 690 nm 19 . This result suggests that the newly formed pigment in the oxidized molischianum-LH2 was not AcChl a. This assumption was confirmed by high-performance liquid chromatography (HPLC) analysis and electronic absorption spectroscopy of the pigments from oxidized molischianum-LH2. Oxidized molischianum-LH2 had two major chlorophyllous pigments (Fig. 3A,B). The former pigment (#1 in Fig. 3A) was BChl a, which we attribute to residual B850 BChl a (Fig. 3C). We assigned the latter pigment (#2 in Fig. 3B) to the oxidized pigment. This oxidized pigment eluted more slowly in the HPLC analysis than AcChl a (Fig. 3D). In addition, the Q y peak position of the oxidized pigment at 692 nm in methanol was red-shifted compared with that of AcChl a at 685 nm (Fig. S5). The oxidized pigment had a signal at m/z 909.5378 in high-resolution mass-spectrometry (HRMS) measurements. This value corresponds to that of the calculated value (MH + , 909.5375) of a didehydrogenated pigment derived from BChl a. These results suggest that the pigment formed through the DDQ oxidation of molischianum-LH2 is an isomer of AcChl a.
To assign the oxidized pigment formed in molischianum-LH2, the pigment was demetallated and compared with a structurally determined 17,18-didehydro-bacteriopheophytin (BPhe) a that was synthesized from BPhe a by oxidation with FeCl 3 11,12 (see "Materials and methods" as well as Fig. S6). Note that the removal of Mg from BChl a is necessary for synthesis of the B-ring reduced chlorin possessing the 13 2 -methoxycarbonyl group. HPLC analysis revealed that the retention time of the free-base derived from the oxidized pigment in molischianum-LH2 was identical to that of authentic 17,18-didehydro-BPhe a (Fig. 4A,B), but differed from that of the D-ring reduced 3-acetyl-pheophytin a (Fig. 4C). The electronic absorption spectrum of the oxidized pigment in methanol was identical to that of 17,18-didehydro-BPhe a (Fig. 4D). These results indicate that the newly formed pigment in molischianum-LH2 by the DDQ oxidation is 17,18-didehydro-BChl a (hereafter denoted as 1).
No porphyrin-type pigment was detected in the pigments that were extracted from oxidized molischianum-LH2, indicating that the treatment of molischianum-LH2 with DDQ did not oxidize the B-ring of B800 BChl a. No conversion from B800 BChl a to the corresponding porphyrin-type pigment, 3-acetyl-protochlorophyll a, has been reported in the DDQ oxidation of acidophilus-LH2 20 . The regioselective oxidation of B800 BChl a observed here suggests that the polypeptides sterically protect the pyrrole ring of BChl a inside the protein matrix.
We confirmed the effects of the DDQ oxidation on lycopene, a major carotenoid in molischianum-LH2, by HPLC analysis. Lycopene from oxidized molischianum-LH2 was detected at the same retention time as that of native LH2 and a standard sample (Fig. S7), indicating that the lycopene in molischianum-LH2 was unaffected by the DDQ oxidation. The stable peak positions of lycopene in the absorption spectrum of oxidized molischianum-LH2 (Figs. 2 and S2) confirmed that DDQ oxidation had no effect on lycopene.
The effects of the B800 oxidation on the protein structure of molischianum-LH2 were examined by a frequency modulation atomic force microscopy (FM-AFM), size-exclusion chromatography (SEC), and circular dichroism (CD) spectroscopy. The crystallographic structure of LH3 protein 21 indicates that the circular arrangement is conserved even if local interactions of BChl a with polypeptides; namely substitution of amino acid residues that participate in the formation of the BChl-binding pocket is proved to produce no effect on the overall structures of light-harvesting proteins from purple bacteria. In contrast, information on the effect of chemical modification of BChl a bound to LH2 and related proteins on their protein structures is less available. The structural analysis demonstrated here will provide useful information in this regard.
The ring structure of oxidized molischianum-LH2 was clearly visualized by FM-AFM (Fig. 5). This circular arrangement is quite similar to that of native molischianum-LH2 22 . The height profiles in the AFM results ( Fig. S8) indicated the averaged top-to-top distance of oxidized molischianum-LH2 to be 4.7 ± 0.2 nm (average and standard deviation of 16 samples). This value is almost identical to that of native molischianum-LH2 (4.5 ± 0.5 nm) 22 . www.nature.com/scientificreports/ Therefore, no deformation of the LH2 ring structure was induced by the in situ oxidation of B800 BChl a in molischianum-LH2. The elution volume of oxidized molischianum-LH2 in the SEC chromatogram was the same as that of native molischianum-LH2 (Fig. S9), indicating that the protein size was unchanged by the oxidation of B800 BChl a.
In the CD spectrum of oxidized molischianum-LH2, a negative CD signal of B850 BChl a was observed at around 855 nm (Fig. S10C). This signal was similar to that of native molischianum-LH2 ( Fig. S10A) 19,22 , indicating that the orientation and electronic structures of B850 BChl a were not influenced by the B800 oxidation. On oxidation of B800 BChl a, a reverse S-shaped signal for B800 BChl a at around 800 nm disappeared and a new negative CD signal appeared at around 700 nm (Fig. S10C). This negative signal is assigned to 1 in B800 sites. Note that AcChl a reconstituted into the B800 site of molischianum-LH2 barely exhibited CD signal in the Q y region 19 . This difference suggests some disarrangements of reconstituted AcChl a in the B800 site of molischianum-LH2 19 , although the interactions of AcChl a with the surrounding amino acid residues are essentially the same as those of 1 and native B800 BChl a. The negative CD signal in the spectrum of oxidized molischianum-LH2 at around 220 nm (Fig. S10D) closely resembles that of native LH2 (Fig. S10B), indicating that the content of α-helices was unchanged by the B800 oxidation.
Excitation of oxidized molischianum-LH2 at 700 nm produced an emission from B850 BChl a at 860 nm (Fig. S11B, black curve). This emission is in line with the B850 emission by excitation of B800 BChl a in native LH2 (Fig. S11A, black curve). In the excitation spectra of oxidized and native molischianum-LH2, the bands at around 700 and 800 nm were detected, respectively (Fig. S11, red curves). These results indicate that 1 and B800 BChl a function as energy donors in oxidized and native molischianum-LH2, respectively.  www.nature.com/scientificreports/ We used femtosecond transient absorption (TA) spectroscopy to examine the EET dynamics from 1 to B850 BChl a in the oxidized molischianum-LH2 (Fig. 6). Excitation of oxidized molischianum-LH2 at 700 nm produced a negative band with a minimum at 700 nm, which was assigned to superposition of the ground state bleach (GSB) and stimulated emission (SE) of the energy-donating 1 in the B800 site. This negative band subsequently decayed, accompanied by new positive and negative bands at 825 and 855 nm, respectively, indicating EET from 1 to B850 BChl a 19,23,24 .
We obtained two decay-associated spectra (DAS) (Fig. 7) by global analysis of the time dependence of the differential absorbance (ΔAbs) at various wavelengths (Fig. S12). This global analysis is in line with our previous analysis of native molischianum-LH2 and AcChl a-reconstituted molischianum-LH2 19 . The DAS component A with a shorter lifetime (6.8 ps) has a negative band at 700 nm, which corresponds to the decrease of the mixed GSB/SE band of 1, with a pair of 825-nm negative and 855-nm positive bands due to the increase of the positive excited-state absorption band and the negative GSB/SE band of B850 BChl a, respectively. The DAS component B with a longer lifetime (1.2 ns) represents the decay of the excited state of B850 BChl a.
The TA spectroscopy results reveal that 1 can function as an energy donor in oxidized molischianum-LH2. The intracomplex EET dynamics from 1 to B850 BChl a is homogeneous, suggesting that the molecular orientation of 1 in the eight B800 pockets is not affected by the DDQ oxidation. The lifetime of the DAS component A, which originates from the combination of the decay of 1 with the rise of B850 BChl a, indicates that intracomplex EET in oxidized molischianum-LH2 occurs approximately sevenfold slowly relative to that in native LH2 (990 fs) 19 . This slow intracomplex EET is attributed to low spectral overlap between energy-donating 1 and energy-accepting B850 BChl a. The intracomplex EET in oxidized molischianum-LH2 was slightly slower than that in AcChl a-reconstituted LH2 (5.0 ps) 19 despite a slight red-shift of the Q y band of 1 in the B800 site (700 nm) compared with that of AcChl a (690 nm). One possible reason for this result is the difference in the electron delocalization between B-ring reduced and D-ring reduced chlorins. Density functional theory (DFT) calculations indicated that electron density was delocalized over the β-position of the D-ring in the LUMO of derivatives of 1, although no delocalization was found at the β-positions of both the B-and D-rings in the LUMO of AcChl a derivatives 11 . Such a difference in the distribution of the electron densities might lead to slightly slow intracomplex EET in oxidized molischianum-LH2.
To summarize, a regioselective isomer of natural Chls, namely B-ring reduced chlorin, is produced by selective in situ oxidation of B800 BChl a in molischianum-LH2. The B-ring reduced chlorin functions as an antenna pigment in the LH2 protein. Regioselective dehydrogenation of the D-ring of B800 BChl a occurs because of its  20 . This different regioselectivity can be rationalized by the difference in the B800 orientations between the two types of LH2 proteins (Fig. S1), and indicates an importance of the polypeptides, which protect the pyrrole rings in BChl a in the protein matrix. Additionally, the regioselectivity we observe in the protein matrix contrasts with that in preferential dehydrogenation of the B-ring of BChl a derivatives in organic solvents 11,25 . This is the first example of a photoactive B-ring reduced chlorin pigment in photosynthetic proteins. We hope that these findings will be helpful for understanding the roles of photosynthetic cyclic tetrapyrrole pigments on the light-harvesting proteins and wrestling with an open question why nature selects D-reduced Chl pigments.

Materials and methods
Apparatus. Electronic absorption and CD spectra were measured with a spectrophotometer (UV-2600, Shimadzu) and a spectropolarimeter (J-820, JASCO), respectively. Fluorescence emission and excitation spectra were measured with a spectrophotometer (F-7100, Hitachi). HPLC was performed with a pump (LC-20AT, Shimadzu) and detectors (SPD-M20A and SPD-20AV, Shimadzu). SEC was performed with an ÄKTAprime plus system (GE Healthcare). 1 H NMR spectra were measured with an NMR spectrometer (ECA-600, JEOL); chemical shifts were expressed (in ppm) relative to CHCl 3 (7.26) as an internal reference. HRMS measurements were conducted with a spectrometer (micrOTOF II, Bruker) by atmospheric pressure chemical ionization (APCI).
Materials. LH2 protein was isolated from the cultured cells of a purple photosynthetic bacterium Phaeospirillum molischianum DSM120 26 . BChl a was isolated from a purple photosynthetic bacterium Rhodobacter sphaeroides, and was converted to AcChl a and BPhe a by DDQ oxidation 27

Isolation of oxidized pigment 1.
Oxidized molischianum-LH2 was concentrated by ultracentrifugation using Amicon centricon concentrators (50 kDa cutoff, Merck Millipore), followed by evaporation with a smart evaporator (BioChromato) in the dark. The pigment 1 was extracted from the resulting sample with methanol www.nature.com/scientificreports/ and purified by reverse-phase HPLC using a column 5C 18 -AR-II (10 mm i.d. × 250 mm, Nacalai Tesque) with methanol at the flow rate of 1.0 mL/min. VIS (methanol) λ max 692 (relative intensity, Synthesis of 17,18-didehydro-BPhe a. 17,18-Didehydro-BPhe a was synthesized from BPhe a by oxidation with FeCl 3 according to previous reports 11,12 . A nitromethane solution of FeCl 3 ⋅6H 2 O (4.0 eq.) was added to a dichloromethane solution of BPhe a, and the mixed solution was stirred at room temperature in the dark for 5 min. The reaction was quenched by addition of methanol and washed with distilled water. The organic layer was dried over anhydrous Na 2 SO 4 , followed by evaporation under reduced pressure.

Transient absorption spectroscopy.
Femtosecond time-resolved TA spectroscopy was done according to a previous report 19 with a pair of noncollinear optical parametric amplifiers (NOPA) (TOPAS-white, Light-Conversion), pumped by a regeneratively amplified Ti:sapphire laser (Solstice, Spectra-Physics), as light sources. Output of one of the NOPAs was set at 700 nm for excitation of the Q y band of the oxidized pigment in oxidized molischianum-LH2. A prism pair was used to pre-compress the pulses and the pulse duration at the sample position was 16 fs (fwhm), which was measured by the self-diffraction frequency-resolved optical gating (SD-FROG) method. The excitation intensity at the sample position was 20 μW (20 nJ), and the diameter of the focused laser beam was ca. 0.15 mm. The polarization between the pump and probe pulses was set at the magic angle by rotating the polarization of the pump pulse by a Berek compensator (Model 5540, New Focus). White-light supercontinuum (410-930 nm) was generated by focusing the output of another NOPA centered at 1100 nm into a rotating CaF 2 window (thickness: 2 mm) and it was divided into probe and reference pulses. The probe pulse was focused into a rotating sample cell excited by the pump pulse, and the transmitted light was guided into a multichrometer (MSP1000-V, Unisoku). The reference pulse was directly guided into another multichrometer of the same type and the differential absorbance (ΔAbs) of the sample was calculated. The heterodyne-detected optical Kerr effect (HD-OKE) signal between the pump and the probe pulses was obtained by replacing the sample solution in the rotating cell with neat carbon tetrachloride, and the electronic response signal was used to compensate the group velocity dispersion of the TA signal. Oxidized molischianum-LH2 was solubilized in www.nature.com/scientificreports/ 20 mM Tris buffer containing 0.02% DDM (pH 8.0), and the Q y absorbance of B850 BChl a of oxidized molischianum-LH2 was set at ca. 0.8-0.9 with the 2-mm optical length.

Data availability
Data generated or analyzed during the current study are included in this published article and are available from the corresponding author on reasonable request.