Chunhe Xu

Study on the photo-generation of superoxide radicals in Photosystem II with EPR spin trapping techniques

Direct EPR evidence of the photo-generation of superoxide radicals (O2−.) was obtained by using a novel spin trapping probe in spinach Photosystem II (PS II) membrane fragments. The production of O2−. was detected by following the formation of 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) superoxide adducts (DEPMPO-OOH). The inhibition of O2−. formation by 3-(3,4-dichlorophenyl) -1,1-dimethylurea (DCMU) and the 77 K fluorescence spectrum indicated that O2−. were generated from PS II, not from PS I. The inhibition of O2−. formation by DCMU also suggested that O2−. were generated from the QBbinding site, not at a site prior to DCMU blockage. The extrinsic proteins and Mn are very important to eliminate O2−., showing that the oxygen-evolving system is involved in O2−. removal rather than production.

Differentiating the orientations of photosynthetic reaction centers on Au electrodes linked by different bifunctional reagents.

The photosynthetic reaction center (RC) composite film was fabricated by self-assembled monolayers (SAMs) on the Au electrode with two different bifunctional reagents, 4-aminothiophenol (ATP) and 2-mercaptoethylamine (MEA), respectively. The square wave voltametry (SWV), bulk electrolysis and photocurrent test were employed for characterizing the composite film. The dramatic different electrochemical characteristics were observed for the two types of films, which strongly suggested an orientational difference for RC arising from the structural difference between the two bifunctional reagents. For RC-MEA film, three redox peaks which implying electron transfer (ET) between the primary donor (P) and the bacteriopheophytin (Bphe) were observed. While for RC-ATP film, two redox peaks implying ET between the nonheme iron and the primary quinone (Q(A)) were observed. The ET behavior driven by electric field also supported the result that the RC could be linked to the electrode at different sites. The site-specific immobilization approach reported here supplies a method to differentiate the protein orientation.

Photoelectric conversion of photosynthetic reaction center in multilayered films fabricated by layer-by-layer assembly

Abstract Multilayered protein films which contained ordered layers of photosynthetic reaction center (RC) from Rhodobacter Sphaeroides (RS601) were assembled by means of alternate electrostatic adsorption with positively charged poly(diallyldimethylammonium chloride) (PDDA). The assembly of RC was monitored by spectrometry and photocurrent measurement. Linear film growth was observed up to about 20 cycles of adsorption. For the monolayer film, the photocurrent was about 8.5 nA cm −2 . For the multilayered film, the total photocurrent was about 77 nA cm −2 for the 24-layer RC film, while the average photocurrent increment per adsorption cycle was about 3.2 nA cm −2 . The overall light-to-electricity conversion efficiency for a 24-layer film was about eight times higher than that for the monolayer one. The effects of electrode potential and pH on the photocurrent were also measured to illustrate the light-to-electric converting mechanism.

Photoelectrochemistry of photosynthetic reaction centers embedded in Al2O3 gel

Abstract Bacterial photosynthetic reaction center (RC) from Rhodobacter sphaeroides strain RS601 was immobilized on the glassy carbon electrode, and its photoelectrochemical responses were investigated. The effects of the H 2 O/Al ratio, protein concentration and pH of the electrolyte on photocurrent were measured. The results showed that: (1) the H 2 O/Al ratio affected the pore size of sol–gel matrix, resulting in the maximum of photocurrent and peak shape changing; (2) the photocurrent increased with the load amount of protein; (3) the optimal pH range for photocurrent was in neutral pH region. The absorption and circular dichroism (CD) spectra and photocurrent results showed that the structure and activity of protein were kept in Al 2 O 3 gel films. This work supplied a promising approach to fabricate artificial biomimic solar cell.

Relationship among Photosystem II carbonic anhydrase, extrinsic polypeptides and manganese cluster

Effects of Photosystem II (PS II) extrinsic polypeptides of oxygen-evolving complex and manganese clusters on PS II carbonic anhydrase (CA) were studied with spinach PS II membranes. The result supported that membrane-bound CA is located in the donor side of PS II. The extrinsic polypeptides played an important role of maintaining CA activity. After removing manganese clusters, oxygen evolution activity was inhibited, but PS II-CA activity was unchanged. It was concluded that CA activity is independent of the presence of manganese clusters, and was not directly correlated with oxygen evolution activity.

PROBING ELECTRON TRANSFER OF THE REDOX SPECIES IN WILD-TYPE RC PROTEIN AND ITS PIGMENT-REPLACED MUTANTS RE-CONSTITUTED IN SELF-ASSEMBLY MONOLAYERS

The multi-layered protein film, containing photosynthetic reaction center (RC) protein isolated from Rhodobacter sphaeriodes or its pigment-replaced mutants, was prepared with 4-Aminothiophenol (4-ATP) self-assembled monolayer (SAM) modified gold electrode. The RC protein was embedded in an ordered-orientated charged poly-styrenesulfonate (PSS) and poly(dimerthyldiallylammonium) chloride (PDDA) film successively. The reversible or quasi-reversible electron transfer (ET) for the re-constituted RC and its mutants was probed by cyclic voltammetry (CV) and square wave voltammetry (SWV), reflecting the electrochemical-driven electron recombination for RC protein or its mutants. The SWV data for RC and its mutants in the films were non-linearly fitted to get the electrochemical parameters.

Replacement of bacteriopheophytin in reaction centers from Rhodobacter sphaeroides RS601 with plant pheophytin

In the presence of acetone and an excess of exogenous plant pheophytins, bacterio-pheophytins in the reaction centers fromRhodobacter sphaeroides RS601 were replaced by pheophytins at sites HA and HB, when incubated at 43.5°C for more than 15 min. The substitution of bacteriopheophytins in the reaction centers was 50% and 71% with incubation of 15 and 60 min, respectively. In the absorption spectra of pheophytin-replaced reaction centers (Phe RCs), bands assigned to the transition moments QX (537 nm) and QY (758 nm) of bacteriopheophytin disappeared, and three distinct bands assigned to the transition moments QX (509/542 nm) and QY (674 nm) of pheophytin appeared instead. Compared to that of the control reaction centers, the photochemical activities of Phe RCs are 78% and 71% of control, with the incubation time of 15 and 60 min. Differences might exist between the redox properties of Phe RC and of native reaction centers, but the substitution is significant, and the new system is available for further studies.

Acquirement and characterization of a carotenoid mutant (GM309) of Rhodobacter sphaeroides 601

A green mutant was obtained among the chemically induced mutants of Rhodobacter sphaeroides 601 (RS601) and named GM309. A blue shift of 20 nm of the carotenoid absorption spectrum was found in the light-harvesting complex II (LH2) of GM309. Different from LH2 of RS601, it was found that the carotenoids in GM309-LH2 changed to be neurosporene by mutation. Neurosporene lacks a conjugate double bond, compared with the spheroidene in RS601-LH2 which has ten conjugate double bonds. As shown by absorption and circular dichroism spectroscopy, the overall structure of GM309-LH2 is little affected by this change. From fluorescence emission spectra, it is found that GM309-LH2 can transfer energy from carotenoids to Bchl-B850 without any change in efficiency. But the efficiency of energy transfer from B800 to B850 in GM309-LH2 is decreased to be 42% of that of the native. This work would provide a novel method to investigate the mechanism of excitation energy transfer in LH2.

Influence of pigment substitution on the electrochemical properties ofRhodobacter sphaeroides 601 reaction centers

With the help of pigment substitution, self-assembled monolayer film and square wave voltammetry, the influence of pigment substitution on the electrochemical properties ofRhodobacter sphaeroides 601 reaction centers was investigated. Results showed that the charge separation could also be driven by externally electric field, similar to the primary photochemical reaction in purple bacterial reaction center. On the surface of Au electrode, a self-assembled monolayer film (the RC-PDDA-DMSA film) was made up of 2,3-dimercaptosuccinic acid (DMSA), poly-dimethyldiallylammonium chloride (PDDA) and reaction center (RC). When square wave voltammetry was used to study the RC-PDDA-DMSA film, four redox pairs in the photochemical reaction of RC were observed by changing frequency. With nonlinear fitting, the standard potential of P/P+ and the corresponding electrode reaction rate constant were determined to be 0.522 V and 13.04 S-1, respectively. It was found that the redox peak at −0.02 V changed greatly when bacteriopheophytin was substituted by plant pheophytin in the reaction center. Further studies indicated that this change resulted from the decrease in electron transfer rate between Bphe-/Bphe (Phe-/Phe) and QA-/QA after pigment substitution. After investigations of spectra and electrochemical properties of different RCs and comparisons of different function groups of pigments, it was indicated that the phytyl tail, similar to other substituted groups of pheophytin, affected the efficiencies of pigment substitution.

On Differences in Accessibility and Redox Activity of D1-Y161 and D2-Y160 of PSII as Probed by Halogenated Acetates

Photosystem II (PSII) includes at least seven intrinsic polypeptides (D1, D2, the α and β subunits of cytochrome b-559, CP47, CP43, and the psbI gene product) and three extrinsic polypeptides with molecular masses of 33, 23, 17 kDa as the key components of this photosystem. A new method has been set up to release sequentially PSII extrinsic polypeptides. When PSII particles were incubated with increasing concentrations of trichloroacetate (TCA), first 17, then 23 and finally 33 kDa polypeptide was released (1). In current research, we have studied how glycinebetaine, a compatible solute, selectively stabilizes the extrinsic polypeptides when PSII particles are treated by different methods. EPR signal measurement displayed that after the removal of the 33 kDa extrinsic polypeptide, small hydrophobic molecules, like halogenated acetates, can access and affect the redox activity of D2-Y160 (2). The model for the microenviroment around D1-Y161 and D2-Y160 are also provided (3).