The following is an abridged version of my research with the most detail pertaining to recent advances by the undergraduates here at Susquehanna University. More background can be found in my CRC review, which also contains pertinent references in the literature. A useful chapter covering the photosynthetic light reactions (general reference) was recently written by Dr. John Golbeck. Several useful references are also given at the end of this page.

Photosynthesis is an important process that converts solar energy into chemical energy, thereby fueling the living world directly or indirectly. In oxygenic photosynthesis, cyanobacteria and the chloroplasts of higher plants and algae sequester light through photosynthesis to release oxygen from water and produce carbohydrates from carbon dioxide reduction. In the light driven reactions of photosynthesis, photosystem I (PSI) and photosystem II (PSII) capture photons to utilize the energy for electron transfer from water to NADP⁺ through three membrane bound complexes: PSII, cytochrome b₆f and PSI. This process produces NADPH and a proton gradient across the thylakoid membranes that is used by ATP synthase to produce ATP. These products, ATP and NADPH, are used to reduce carbon dioxide in the production of carbohydrates (sugars).

From Voet and Voet, Biochemistry, 3rd Edition.

Photosystem I (PSI) is a well-characterized photosynthetic reaction center in cyanobacteria and higher plants. PSI catalyzes the photooxidation of plastocyanin in the thylakoid lumen and the photoreduction of ferredoxin in the cyanobacterial cytoplasm (or chloroplast stroma). PSI contains at least eleven proteins, with PsaA and PsaB as a heterodimeric core. Surrounding PsaA/PsaB are 6 small subunit proteins, that serve various identified or unknown functions. In addition to light-harvesting chlorophyll a (Chl a) molecules, PsaA/PsaB is imbedded with the P700 reaction center and a series of cofactors (A₀, A₁, F_X) that act as intermediate electron acceptors and provide a charge separation from the initial P700 reaction center. P700 is comprised of a dimer of Chl a molecules. The electron acceptor cofactors include a molecule of chlorophyll a, A₀, phylloquinone, A₁ and a [4Fe-4S] iron sulfur protein, F_X. PSI also contains three overlapping peripheral proteins (PsaC, PsaD and PsaE). PsaC is a prerequisite for PsaD and PsaE binding and contains the two terminal electron transfer [4Fe-4S] iron sulfur clusters F_A and F_B . F_A and F_B act as an electron shuttle from the PsaA/PsaB core cofactors to docked ferredoxin. PsaD provides a ferredoxin docking site, with PsaE assisting in the interaction of PSI with ferredoxin .

All well-characterized photosynthetic reaction centers contain a bound quinone molecule. Type II reaction centers, PS II, contain two plastoquinone: one that functions as a bound secondary electron acceptor and the other that functions as mobile terminal electron acceptor. Type I reaction centers, such as PS I, contain two bound phylloquinone (PhQ, vitamin K₁, 2-methyl-3-phytyl-1,4-naphthoquinone, labeled Q_K-A and Q_K-B, above). Phylloquinone becomes neither protonated as part of the electron transfer process nor diffuses from the PSI complex as part of its normal function. This molecule acts as an intermediate in the electron transfer from the primary acceptor A₀, a chlorophyll a to F_X, an iron-sulfur cluster. PhQ acts as both a physical and energetic bridge for the electron between the primary acceptor and the terminal iron sulfur clusters. Phylloquinone plays an important role as a participant in the early stages of electron transfer in PS I. Yet the structure function relationship of phylloquinone in PSI is not fully understood. The primary objective of this research is to probe the interaction of quinone like molecules in PS I. Specifically, we will incorporate foreign quinones into PSI and characterize the physiological and bioenergetic aspects of PS I. The system of study is a Synechocystis sp PCC 6803.

Dr. Johnson mutationally disabled several of the genes (menA, menB, menD, menE) in the phylloquinone biosynthetic pathway. Two mutants of note are menA (phytyl transferase) and menB (DHNA synthase). The menA mutant is incapable of attaching the phytyl tail to the quinone, which is important for successful quinone incorporation into PS I. The menB mutant prevents the second ring closure, but allows the phytyl transfer to occur. These mutant cells were shown to lack phylloquinone and the ability to grow at high light. All of the men mutants(menA, menB, menD, menE) utilize plastoquinone as an alternative to phylloquinone. However, they were shown to re-incorporate phylloquinone into PS I by simple addition to the growth medium. The menB mutant has the capacity to modify quinone-like molecules in vivo and to incorporate them intact into PS I. Only phytylated quinones grow at high light. The full extent of the incorporation of these quinone-like molecules in to PS I has not been adequately explored. The CRC Review provides greater detail on the physiological and energetic changes in the mutants.

The focus this research on the menA and menB mutants (menB18), which will not grow at high light levels. Previously, the Golbeck laboratory showed that a series of anthraquinones added to the growth medium allows the menB18 cells to grow at high light (Figure 1). Surprisingly, the menA mutant with anthraquinones added to the growth media has been found to grow at high light (Figure 2). The growth rates approach that of wild type for both the menA and menB18 cells supplemented with anthraquinones. The implication is that the phytyl tail is not attached, and the the unmodified anthraquinones are incorporated into the quinone binding site of PS I. This is the first example of a non-tailed quinone acting as a secondary electron acceptor in vivo.

Figure 1. Log phase menB18 mutant grown at high light (120 mE) supplemented with 9,10-anthraquinones (AQ): no added quinone; AQ; 1-CH 3NH 2-AQ; 2,3-(CH 3) 2-AQ; 1-NH 2-AQ.

Figure 2. Log phase menA mutant grown at high light (120 mE) supplemented with 9,10-anthraquinones (AQ): no added quinone; AQ; 1-CH3NH 2-AQ; 2,3-(CH3) 2-AQ; 1-NH2-AQ.

Wild type and menB (and menA) show back reaction times of 100 ms and 3 ms, respectively (Figure 3). The menB mutant supplemented with 1-NH2-CH3-9,10-AQ shows a back reaction time of 63 ms (the kinetic major phase). Transient EPR studies (Figure 4) are also consistent with the in vitro incorporation of 9,10-anthraquinone 3. This experiment was performed at Pennsylvania State University in Dr. John Golbeck's Lab. Professor Golbeck is a collaborator on this research and has allowed us the use of his instruments. Several of instrumental set ups are home built or highly specialized. The kinetic back reaction experiment uses two lasers to monitor the rereduction of the P700+ cofactor. The excitation pump pulses a 532 nm (green) beam which excites the PSI complex. The probe laser at 811 nm (deep red) monitors the reduction of P700+ as a function of time.

Spin polarized transient X-band EPR is an advanced technique performed at Brock University (Ontario, Canada) by Dr. van der Est. The analysis of the data shows that the anthraquinones added in vivo to the growth culture incorporate into PSI. The in vivo menB + methyl amino-AQ spectrum is comparable to the wild type in vitro experiment where the AQ was added to PSI through chemical means (Figure 4). Note that the wild type and menb spectra are distinctly different. This is supporting evidence that AQ is properly incorporated into PSI and functions like a native quinone.

References

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