Takumi Noguchi

The process of formation of the triplet state of chlorophyll in the photosystem II (PS II) reaction center complex was studied by means of time-resolved infrared (IR) spectroscopy. Using a dispersive-type IR spectrometer with a time resolution of ∼55 ns, transient spectra in the CO stretching region (17601600 cm^-1) were measured at 77 K. The data were analyzed by singular-value decomposition and subsequent least-squares fitting. Two distinct spectral components having different kinetic behaviors were resolved. One had spectral features characterized by negative peaks at 1740 and 1680 cm^-1 and an overall positive background and was assigned to the P₆₈₀⁺Phe^-/P₆₈₀Phe radical pair by static FTIR measurements of the P₆₈₀⁺/P₆₈₀ and Phe^-/Phe differences. The other had prominent negative and positive peaks at 1668 and 1628 cm^-1, respectively, which were previously assigned to the keto CO change upon triplet formation of the monomeric chlorophyll denoted as Chl_T [Noguchi, T., Tomo, T., and Inoue, Y. (1998) Biochemistry 37, 13614−13625]. The former component of P₆₈₀⁺Phe^-/P₆₈₀Phe exhibited a multiphasic decay with time constants of 77 ns (75%), 640 ns (18%), 8.3 μs (4%), and 0.3 ms (3%), while the latter component of ³Chl_T/Chl_T was formed with a single-exponential rise with a time constant of 57 ns and had a lifetime of 1.5 ms. From the observations that only the two spectral components were resolved without any other triplet intermediates and the time constant of ³Chl_T formation roughly agreed with or seemed even faster than that of the major phase of the P₆₈₀⁺Phe^- decay, two alternative mechanisms of triplet formation are proposed. (i) ³Chl_T is directly formed from P₆₈₀⁺Phe^- by charge recombination at Chl_T, and (ii) ³P₆₈₀ is formed, and then the triplet is transferred to Chl_T with a time constant of much less than 50 ns. The location of Chl_T in the D1 subunit as the monomer chlorophyll corresponding to the accessory bacteriochlorophyll in the L subunit of purple bacteria is favored to explain the former mechanism as well as the triplet properties reported in the literature. The physiological role of the triplet formation on Chl_T is also discussed.

Fourier transform infrared (FTIR) difference spectra of all flash-induced S-state transitions of the oxygen-evolving complex were measured using photosystem II (PSII) core complexes of Synechococcus elongatus. The PSII core sample was given eight successive flashes with 1 s intervals at 10 °C, and FTIR difference spectra upon individual flashes were measured. The obtained difference spectra upon the first to fourth flashes showed considerably different spectral features from each other, whereas the fifth, sixth, seventh, and eighth flash spectra were similar to the first, second, third, and fourth flash spectra, respectively. The intensities at the wavenumbers of prominent peaks of the first and second flash spectra showed clear period four oscillation patterns. These oscillation patterns were well fitted with the Kok model with 13% misses. These results indicate that the first, second, third, and fourth flash spectra represent the difference spectra upon the S₁ → S₂, S₂ → S₃, S₃ → S₀, and S₀ → S₁ transitions, respectively. In these spectra, prominent bands were observed in the symmetric (1300−1450 cm^-1) and asymmetric (1500−1600 cm^-1) stretching regions of carboxylate groups and in the amide I region (1600−1700 cm^-1). Comparison of the band features suggests that the drastic coordination changes of carboxylate groups and the protein conformational changes in the S₁ → S₂ and S₂ → S₃ transitions are reversed in the S₃ → S₀ and S₀ → S₁ transitions. The flash-induced FTIR measurements during the S-state cycle will be a promising method to investigate the detailed molecular mechanism of photosynthetic oxygen evolution.

A water-soluble Chl a/b-protein complex, CP668, from Chenopodium album converts to another form of protein complex, CP743, upon light illumination. Structural changes of pigments and proteins upon photoconversion were studied using resonance Raman (RR) and Fourier transform infrared (FTIR) spectroscopies. RR spectra of CP668 and CP743 and a light-induced FTIR difference spectrum showed that the macrocyle C=C bands of Chl a in CP668 considerably changed upon conversion to the pigment (not chemically identified yet) in CP743. The C=C band pattern of the RR spectrum of CP743 was similar to that of bacteriochlorophyll a, suggesting that the conjugated system of the CP743 pigment resembles a bacteriochlorin ring. Judging from the C=O frequencies, the 13¹-keto C=O groups of Chl a and b in CP668 are free from hydrogen bonding, whereas the 13²-ester C=O groups of both Chl a and b and the 7-formyl C=O of Chl b in CP668 are hydrogen bonded. Upon conversion to CP743, interactions of the 13¹-keto and 13²-ester C=O groups were basically unaffected, demonstrating no drastic changes around these C=O groups. FTIR spectra in the amide I' region of CP668 and CP743 in D₂O buffer showed a peak at 1,633 cm⁻¹, which represents a major component of β-sheet conformation. Second-derivative spectra of the amide I' bands as well as a light-induced FTIR difference spectrum suggested that drastic change in the protein conformation does not occur upon photoconversion.

A Fourier transform infrared (FTIR) difference spectrum of the primary electron donor (P680) of photosystem II upon its photooxidation (P680⁺/P680) was obtained in the frequency region of 1000−3000 cm^-1. The reaction center (RC) complex (D1−D2−Cytb559) was used for the measurements in the presence of ferricyanide as an exogenous electron acceptor. Control measurements of electronic absorption (300−1200 nm) showed that illumination of the RC complex at 150 K induced major oxidation of P680 concomitant with oxidation of a carotenoid and an accessory chlorophyll (Chl). Illumination at 250 K also specifically bleached one of the two β-carotene molecules bound to the RC complex, and the sample thus treated exhibited little formation of a carotenoid cation on subsequent illumination at 150 K. The P680⁺/P680 FTIR difference spectrum (with minor contamination of Chl⁺/Chl) was measured at 150 K using this partially carotenoid-deficient RC complex. The spectrum showed a broad positive band centered at ∼1940 cm^-1, which could be ascribed to an infrared electronic transition of P680⁺ analogous to that previously observed in various bacterial P⁺. This finding indicates that a positive charge is delocalized over (or hopping between) the two Chl molecules in P680⁺. The low intensity of this electronic band compared with that of the bacterial band could have three possible explanations: weak resonance interaction between the constituent Chl molecules, an asymmetric structure of P680⁺, and the difference in Chl species. Bands in the CO stretching region (1600−1750 cm^-1) were interpreted in comparison with resonance Raman spectra of the RC complex. The negative peaks at 1704 and 1679 cm^-1 were proposed as candidates for the keto C₉O bands of P680. The observation that neither of these bands agreed with the main keto C₉O band at 1669 cm^-1 in the previous ³P680/P680 FTIR spectrum [Noguchi et al. (1993) Biochemistry 32, 7186−7195] led to the idea that the triplet state migrates to a Chl (designated as Chl_T) different from P680 at low temperatures. Based on these results, structural models of Chl molecules including P680 and Chl_T and their coupling in the cation, triplet, and Qy singlet states are discussed.

Fourier transfer infrared (FTIR) signals originating from a high-potential electron acceptor in PS II were studied by flash-induced FTIR difference spectroscopy. Redox titration of these signals using ferri-ferrocyanide mixtures showed midpoint potentials (_m of 489± 12 and 426±9 mV at pH 5.5 and 6.5, respectively, revealing a pH dependence of about-60 mV/pH unit. These E_m values and pH dependence were in good agreement with those of the non-heme iron, so-called Q₄₀₀, * located between Q^A and Q^B. This indicates that the observed FTIR signals are due to changes in ligands of the non-heme iron and surrounding protein moieties induced on photoreduction from Fe³ to Fe^2-.