Masayo Iwaki

Structural basis for dynamic mechanism of proton-coupled symport by the peptide transporter POT

Proton-dependent oligopeptide transporters (POTs) are major facilitator superfamily (MFS) proteins that mediate the uptake of peptides and peptide-like molecules, using the inwardly directed H+ gradient across the membrane. The human POT family transporter peptide transporter 1 is present in the brush border membrane of the small intestine and is involved in the uptake of nutrient peptides and drug molecules such as β-lactam antibiotics. Although previous studies have provided insight into the overall structure of the POT family transporters, the question of how transport is coupled to both peptide and H+ binding remains unanswered. Here we report the high-resolution crystal structures of a bacterial POT family transporter, including its complex with a dipeptide analog, alafosfalin. These structures revealed the key mechanistic and functional roles for a conserved glutamate residue (Glu310) in the peptide binding site. Integrated structural, biochemical, and computational analyses suggested a mechanism for H+-coupled peptide symport in which protonated Glu310 first binds the carboxyl group of the peptide substrate. The deprotonation of Glu310 in the inward open state triggers the release of the bound peptide toward the intracellular space and salt bridge formation between Glu310 and Arg43 to induce the state transition to the occluded conformation.

Extraction of vitamin K-1 from Photosystem I particles by treatment with diethyl ether and its effects on the A−1 EPR signal and System I photochemistry

Treatment of Photosystem I particles, prepared by digitonin treatment of spinach chloroplasts, with dry diethyl ether extracted all the vitamin K-1 (2.2 molecules/P-700 in the original Photosystem I particles) and carotenoids as well as 60% of antenna chlorophylls. P-700 and FeS centers (FX, FA and FB) were not extracted, and were photochemically oxidized and reduced, respectively, upon continuous illumination. This preparation showed the A−0 EPR signal but no A−1 signal even after long illumination at 210 or 230 K, in which condition A−1 was accumulated in the untreated Photosystem I particles. Laser flash excitation induced only a small absorption change, corresponding to 20% of the total P-700, at 429 nm in the extracted preparation, suggesting that charge recombination took place on a submicrosecond time scale. Addition of vitamin K-1 or K-3 but not that of benzyl viologen increased the extent of the slow decay phase of P-700+. The extraction also induced a new fluorescence band peaking at 692 nm, the intensity of which increased upon addition of dithionite. This fluroescence band is possibly related to the rapid charge recombination between A−0 and P-700+. These results strongly suggest that A1 is vitamin K-1 and that it functions to mediate electron transfer between A0 and FeS centers. Similar results were also obtained with the ‘P-700 enriched particles’ with 7–16 antenna chlorophyll aP-700 which can be obtained by extraction of Photosystem I particles with diethyl ether containing (80% saturated) water.

Modification of photosystem I reaction center by the extraction and exchange of chlorophylls and quinones.

The photosystem (PS) I photosynthetic reaction center was modified thorough the selective extraction and exchange of chlorophylls and quinones. Extraction of lyophilized photosystem I complex with diethyl ether depleted more than 90% chlorophyll (Chl) molecules bound to the complex, preserving the photochemical electron transfer activity from the primary electron donor P700 to the acceptor chlorophyll A(0). The treatment extracted all the carotenoids and the secondary acceptor phylloquinone (A(1)), and produced a PS I reaction center that contains nine molecules of Chls including P700 and A(0), and three Fe-S clusters (F(X), F(A) and F(B)). The ether-extracted PS I complex showed fast electron transfer from P700 to A(0) as it is, and to FeS clusters if phylloquinone or an appropriate artificial quinone was reconstituted as A(1). The ether-extracted PS I enabled accurate detection of the primary photoreactions with little disturbance from the absorbance changes of the bulk pigments. The quinone reconstitution created the new reactions between the artificial cofactors and the intrinsic components with altered energy gaps. We review the studies done in the ether-extracted PS I complex including chlorophyll forms of the core moiety of PS I, fluorescence of P700, reaction rate between A(0) and reconstituted A(1), and the fast electron transfer from P700 to A(0). Natural exchange of chlorophyll a to 710-740 nm absorbing chlorophyll d in PS I of the newly found cyanobacteria-like organism Acaryochloris marina was also reviewed. Based on the results of exchange studies in different systems, designs of photosynthetic reaction centers are discussed.

Two molecules of cytochrome c function as the electron donors to P840 in the reaction center complex isolated from a green sulfur bacterium, Chlorobium tepidum

A photoactive reaction center complex was isolated from a thermophilic green sulfur bacterium, Chlorobium tepidum under anaerobic conditions. The electron transfer occurred from heme c to the photo‐oxidized reaction center chlorophyll, P840+, with a half time ( ) of 110 or 340 μs at 24 or 12°C, respectively. Optical measurements under multiflash excitations indicated that two hemes function as the immediate electron donors to P840+. SDS‐PAGE analysis of the RC complex in combination with the N‐terminal amino acid sequence analyses revealed five subunit bands; a core protein (65 kDa), the light harvesting bacteriochlorophyll a protein (41 kDa), a protein with 2[4Fe‐4S] clusters (31 kDa), monoheme cytochrome c (22 kDa), and a 18‐kDa protein whose function is unknown. The reaction center complex, thus, contains two molecules of cytochrome c per P840.

Electron spin echo of spin-polarised radical pairs in intact and quinone-reconstituted plant photosystem I reaction centers

Abstract Light-induced spin-polarised P700+A1− pairs in intact and quinone-reconstituted photosystem I reaction centres were studied by electron spin echo (ESE) spectroscopy. The observed strong ESE envelope modulation was attributed to magnetic dipolar and exchange interactions in the pairs. The values of these interactions were derived from Fourier-transformed time traces and appeared to be D = −1.71 ± 0.05 G and J = 0.010 ± 0.015 G, respectively. A magnetic field effect on the radical pair lifetime induced by microwave pumping was observed. The reconstituted 2,3-dibromo-1,4-naphthoquinone was shown to be located in the same (A1) site as the native phylloquinone.

Electron transfer in spinach photosystem I reaction center containing benzo-, naphtho- and anthraquinones in place of phylloquinone

Quinone; Phylloquinone; Vitamin K1; Photosystem I; Reaction center; Electron transfer; Photosynthesis

Vitamin K1 (phylloquinone) restores the turnover of FeS centers in the ether‐extracted spinach PS I particles

The effects of vitamin K1 (phylloquinone) addition on the turnover of the FeS centers in photosysten, I photochemistry were studied in diethyl ether‐extracted spinach photosystem I particles. Reconstitution of one Molecule of vitamin K1/reaction center was sufficient to suppress the charge recombination between the oxidized reaction center chlorophyll P700+ and the reduced electron acceptor intermediate chlorophyll a and to restore the turnover of FeS centers in the ether‐extracted particles. This strongly suggests that reconstituted vitamin K1, functions the primary electron acceptor A1 and exhibits a redox midpoint potential low enough to reduce the FeS centers. The quinone binding site in photosystem I, which enables vitamin K1 to show such a low redox potential, seems to be more hydrophobic than those in reaction centers of photosystem II or purple bacteria.

Methods to probe protein transitions with ATR infrared spectroscopy

We describe techniques that can be used in conjunction with modern attenuated total reflection (ATR) infrared micro-prisms to allow proteins to be manipulated cyclically between different states whilst simultaneously monitoring both mid-IR and UV/visible/near IR changes. These methods provide increased flexibility of the types of changes that can be induced in proteins in comparison to transmission methods. Quantitative measurements can be made of vibrational changes associated with conversion between stable catalytic reaction intermediates, ligand binding and oxidation-reduction. Both hydrophobic and soluble proteins can be analysed and the ability to induce transitions repetitively allows IR difference spectra to be acquired at a signal/noise sufficient to resolve changes due to specific cofactors or amino acids. Such spectra can often be interpreted at the atomic level by standard IR methods of comparisons with model compounds, by isotope and mutation effects and, increasingly, by ab initio simulations. Combination of such analyses with atomic 3D structural models derived from X-ray and NMR studies can lead to a deeper understanding of molecular mechanisms of enzymatic reactions.

Topology of pigments in the isolated Photosystem II reaction center studied by selective extraction

Abstract Pigments in the purified spinach Photosystem II reaction center (D1-D2-Cyt b-559) complex were extracted with diethyl ether containing varied amounts of water. The purified reaction center originally contained approximately six molecules of chlorophyll a, two β-carotene and two pheophytin a per one photochemically active pheophytin a. The treatment with 30–50% water-saturated ether extracted one β-carotene, as well as one chlorophyll a that absorbs at 677 nm, remaining 62% of the photochemical activity to reduce pheophytin a. With 60–80% water-saturated ether, almost all the β-carotenes were extracted, remaining the 49% activity without additional loss of chlorophyll. The absorption, fluorescence excitation and linear dichroism spectra demonstrated two spectral forms of β-carotene. The short-wavelength form of β-carotene with absorption peaks at 429, 458 and 489 nm was selectively extracted with ether at low water content, whereas the long-wavelength form with peaks at 443, 473 and 507 nm was extractable only at the higher water content. The extraction enhanced the photobleaching of chlorophylls. The results suggest that chlorophyll a forms with peaks at 667 and 675 nm are located close to the short-wavelength form of β-carotene that can transfer excitation energy to the photoactive pheophytin a on the D1 protein.

Photoconversion Mechanism of a Green/Red Photosensory Cyanobacteriochrome AnPixJ: Time-Resolved Optical Spectroscopy and FTIR Analysis of the AnPixJ-GAF2 Domain

The photoconversion mechanism of a green/red sensory cyanobacteriochrome AnPixJ was studied. The phycocyanobilin-binding second GAF domain of AnPixJ of Anabaena sp. PCC 7120 was expressed in Escherichia coli cells. The His-tagged AnPixJ-GAF2 domain exhibited photoconversion between the green- and red-absorbing forms, APg(543) and APr(648), respectively. We detected four intermediate states in the photocycle between them, as follows: APr(648) → red light → APr(648)* → (with a rise time constant τ(r) of <100 ns) R1(650-80) (with a decay time constant τ(d) of <1 μs) → R2(610) (τ(d) = 920 μs) → APg(543) → green light → APg(543)* → (τ(r) < 50 ns) G1(570) (τ(d) = 190 μs) → G2(630) (τ(d) = 1.01 ms) → APr(648). These intermediates were named for their absorption peak wavelengths, which were estimated on the basis of the time-resolved difference spectra and global analysis of the time courses. The absorption spectrum of APr(648) resembles that of the Pr form of the phytochrome, while all the other states showed peaks at 530-650 nm and had wider bandwidths with smaller peak amplitudes. The fastest decay phases of fluorescence from APr(648)* and APg(543)* gave lifetimes of 200 and 42 ps, respectively, suggesting fast primary reactions. The APg(543)-minus-APr(648) difference FTIR spectrum in an H(2)O medium was significantly different from those reported for the Pfr/Pr difference spectra in phytochromes. Most of the peaks in the difference spectrum were shifted in the D(2)O medium, suggesting the high accessibility to the aqueous phase. The interactions of the phycocyanobilin chromophore with the surrounding amino acid residues, which are fairly different from those in the GAF domain of phytochromes, realize the unique green/red photocycle of AnPixJ.