(2) (25%) Photosystem II (PSII) is an oxidoreductase found in the thylakoid membrane of organisms that perform oxygenic photosynthesis. The fully assembled PSII complex consists of approximately 20 protein subunits and multiple redox-active cofactors that allow PSII to function as a catalyst for the light-driven oxidation of water and concomitant reduction of plastoquinone (1-4). This conversion of sunlight to chemical energy initiates the photosynthetic electron transport chain and sustains nearly all life on Earh.
The active site for water oxidation, a Mn4CaO5 cluster (henceforth Mn cluster), is buried within the PSlI complex near the lumen-membrane interface (5). This buried position limits water access to the active site, a key feature that promotes the reaction. When PSII is modified to allow an unrestricted flow of water to the active site, incomplete water oxidation occurs, forming hydrogen peroxide (H202) that is
reduced to the harmful hydroxyl radical (HO.) (6-9). The buried active site minimizes, but does not completely prevent, such a side reaction pathway, allowing some reactive oxygen species (ROS) to be produced under physiological conditions (10, 11). ROS-induced damage to PSII is the major mechanism believed to be responsible for the frequent PSII turnover that occurs in cells (2, 12), and oxidative modifications of PSII residues near the Mn cluster have been detected (11, 13-17).
Although PSII is the only known enzyme capable of complete water oxidation, it is not the only enzyme for which water is more than just a solvent. Aquaporins transport water across membranes; cytochrome c oxidases produce water as a by-product while pumping protons; and the haloalkane dehalogenases use specific interactions with carefully placed internal water molccules to achieve catalysis. For these and many other enzymes, it is becoming increasingly clear that water dynamics is exquisitely tuned by the protein scaffold to fit the enizyme's function. For example, specific channels regulate the accessibility, geometrical positioning, and flow rate of water molecules within the abovementioned enzymes and in many others (18-21).
Water transport within PSII is of prime interest, given water's unprecedented role as substrate and the problems associated with unrestricted water access to the active site. A number of computational studies have searched for water channels by examining cavities within the static PSII crystal structure (22-24) or by performing molecular dynamics (MD) simulations (25-30). Pathways for removal of the reaction products, dioxygen and protons, have also been considered (22-24, 27. 28, 30) because the extended presenc ce of dioxygen can lead to protein damage (from conversion to singlet dioxygen) (31), and of protons, to inhibition of catalysis (due to improper redox leveling) (32,33). These computational studies have identified several putative channel systems within PSII.
Here, we took an experimental approach to identify oxidized residues on the lumenal side of PSII from the cyanobacterium Synechocystis sp. PCC 6803 (henceforth Synechocystis) by using high-res solution tandem mass spectrometry (MS). We reasoned that after generation near the Mn cluster, ROS would diffuse through putative channels that lead away from the cluster and modify most readily the residues that line the walls of these channels. The app proach is related to a typical hydroxyI radical footprinting experiment, in which hydroxyI radicals are generaled from solvent water molecules by an x-ray or laser pulse (34-37) or via a metal-catalyzed reaction (36, 38-40) and then modify nearby residues. The modified residues, detected by MS, leave a trai! of oxidative damage that, when identified, can
illuminate structural aspects of the system being studied. Hence, the ROS produced at the Mn cluster serve as a built-in footprinting reagent. Using this approach, we identified three nearly continuous formations of oxidized residues that are centered at the Mn cluster and radiate outward all the way to the bulk solvent. Hydrogen peroxide and the hydroxyI radical are the two major ROS known to be produced at the Mn cluster (6, 11). The hydroxyl radical, by far the more reactive species, is short-lived but can diffuse several tens of angstroms after generation at a protcin site (38, 41, 42). Given the similar size and hydrophilicity of HOㆍ and water (43), an HOㆍ diffusion pathway is likely to be favorable for water as well. We conclude that the three ROS channels identified in our study represent three possible water channels in PSII. We discuss the implications of our results for the delivery of water to the site of its oxidation in PSII.
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