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Review
. 2013 Nov 26:4:473.
doi: 10.3389/fpls.2013.00473.

Studying the oxidation of water to molecular oxygen in photosynthetic and artificial systems by time-resolved membrane-inlet mass spectrometry

Dmitriy Shevela  1 , Johannes Messinger
Affiliations
Review

Studying the oxidation of water to molecular oxygen in photosynthetic and artificial systems by time-resolved membrane-inlet mass spectrometry

Dmitriy Shevela et al. Front Plant Sci. .

Abstract

Monitoring isotopic compositions of gaseous products (e.g., H2, O2, and CO2) by time-resolved isotope-ratio membrane-inlet mass spectrometry (TR-IR-MIMS) is widely used for kinetic and functional analyses in photosynthesis research. In particular, in combination with isotopic labeling, TR-MIMS became an essential and powerful research tool for the study of the mechanism of photosynthetic water-oxidation to molecular oxygen catalyzed by the water-oxidizing complex of photosystem II. Moreover, recently, the TR-MIMS and (18)O-labeling approach was successfully applied for testing newly developed catalysts for artificial water-splitting and provided important insight about the mechanism and pathways of O2 formation. In this mini-review we summarize these results and provide a brief introduction into key aspects of the TR-MIMS technique and its perspectives for future studies of the enigmatic water-splitting chemistry.

Keywords: O2 evolution; isotope labeling; isotope-ratio membrane-inlet mass spectrometry; photosynthetic and artificial water-splitting; photosystem II; water-oxidizing complex.

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Figures

Figure 1
Figure 1
Cyanobacterial PSII structure and Kok cycle of photosynthetic water oxidation by the Mn4CaO5 cluster. The arrows within PSII indicate the direction of electron transfer which comprises the following redox-active cofactors: inorganic Mn4CaO5 cluster, redox-active tyrosine Z (YZ), the primary electron donor P680 that includes a pair of Chls a (PD1 and PD2) and two accessory Chls (ChlD1 and ChlD2), the primary pheophytin (PheoD1) acceptor, the primary (QA) and the secondary (QB) quinone acceptors. The phytyl tails of the Chl's and Pheo's, and the isoprenyl chains of the quinones have been cut for clarity. The light-induced S state transitions of the Mn4CaO5 cluster are indicated by arrows with “hν” labels. The PSII structure and the zoomed structural model of the Mn4CaO5 cluster in the center of the Kok cycle are based on the recent PSII crystal structure at a resolution of 1.9 Å (PDB entry 3ARC; Umena et al., 2011).
Figure 2
Figure 2
Representation of a TR-IR-MIMS set-up. Gaseous products, produced by sample suspension (for instance, by PSII samples) in the cell, penetrate through a gas-permeable membrane into a high-vacuum space, pass through a cryogenic trap (which removes water vapor from a flow of gaseous analytes), and enter the isotope ratio mass spectrometer. Here, gaseous analytes are first ionized in the ion source by electron impact, and are then separated according to their m/z ratios by a magnetic field in the sector analyzer that allows simultaneous online detection by individual collector cups (e.g., a 7-cup Faraday detector array). MS signals are monitored and analyzed using a personal computer. See text for further details.
Figure 3
Figure 3
TR-MIMS experiments demonstrating that HCO−3 is not a tightly bound ligand to the Mn4CaO5 cluster in spinach PSII membrane fragments. (A) Amount of released CO2 upon formate addition (black arrows) to intact PSII membranes is the same as in the case of PSII membranes without the Mn4CaO5 cluster (due to 75-min pre-incubation with 80 mM N2H4). Due to enrichment of sample suspension with H182O (3%) CO2 was detected as C16O18O at m/z 46. (B) Addition of the strong reductant NH2OH (white arrows) at concentrations known to cause rapid reduction of the Mn4CaO5 cluster and release of Mn ions as MnII into the solution didn't lead to a release of CO2/HCO−3 above background. In order to avoid the overlay of CO2 and N2O signals (the latter is known to be produced during interaction of NH2OH with the Mn4CaO4 cluster), the N2O signal was shifted from m/z 44 to m/z 46 by employing the 15N-labeled NH2OH for these experiments. To facilitate equilibration between CO2 and HCO−3 all measurements were performed in the presence of externally added CA (to a final concentration of 3 μg ml−1). Modified from Shevela et al. (2008b).
Figure 4
Figure 4
Protocol for TR-MIMS measurements of H216O/H182O exchange in the S3 state of PSII (A) and experimentally obtained substrate water exchange rates in spinach thylakoids (B). (A) The S3 state is populated by two pre-flashes given at 2 Hz (shown by the two first black vertical arrows). This is followed by the rapid injection of H182O into the PSII sample (shown by blue vertical arrow) and subsequent fast mixing of the injected H182O with the sample. Evolution of O2 isotopologues is then induced by a 3rd flash, given at varying delay times (from 0 to 10 s) after the H182O injection (signified as incubation time). Finally, a series of four flashes is given at 2 Hz to induce O2 yield used for normalization. (B) TR-MIMS measurements of substrate H216O/H182O exchange kinetics were performed at m/z 34 (top plot) for singly-labeled isotopologue 16O18O, and at m/z 36 (bottom plot) for doubly-labeled 18O18O in the S3 state in spinach thylakoids at 10°C and pH 6.8. Symbols in both plots are experimental data, and the lines in the top and bottom plots are biexponential and monoexponential fits, respectively. The biexponential fit yields rate constants of ~40 s−1 for the fast phase and ~2 s−1 for the slow phase. The slow phase in the 16O18O data is matching the rate found in the monoexponential fit of the 18O18O data (Messinger et al., ; Hillier et al., ; Hillier and Wydrzynski, 2000, 2004). Adapted from Cox and Messinger (2013).
Figure 5
Figure 5
Schematic representation of the pressure cell (A) specially designed for TR-MIMS measurements of light-induced 18O2 evolution of PSII under high 16O2/N2 pressures (up to 20 bars). (B) MIMS signals in panel (B); Left: 18O2 production of PSII core complexes from Synechocystis sp. PCC 6803 induced by a series of 200 saturating Xenon flashes (given at 2 Hz; indicated by arrow) at 21.7 bars O2, or 20 bars N2. Other conditions: 30% H182O enrichment; [Chl] = 50 μM; 250 μM DCBQ, pH 6.7, 20°C. Right: Flash-induced 18O2 evolution patterns of PSII membrane fragments from spinach induced by a series of saturating laser flashes (separated by dark times of 25 s) at 20.4 bars O2, or 20.2 bars N2. Other conditions: as above, but with 40% H182O. Adapted from Shevela et al. (2011a).
Figure 6
Figure 6
Development of the 18O-isotope fraction (18α) over time for the course of the catalytic O2-formation in reactions catalyzed by synthesized CaMn2O4 · H2O oxide (A,B) and by the WOC of PSII (C). (A) Change in 18α-value for the reaction of CaMn2O4 · H2O with HSO−5 (oxone) indicating that only one of the two oxygen atoms of O2 evolved originates from the bulk water. A solution of HSO−5 in H182O-enriched water was injected at t = 0 into the MIMS cell filled in with a non-enriched oxide suspension (1 mg ml−1; pH ~4.5) to give a final HSO−5 concentration of 3.7 mM and an H182O enrichment of 5%. Note that the rise of 18α to the value of 2.5% corresponds to half the percentage of the 18O-labeled water. (B) Change in 18α-value for the reaction of CaMn2O4 · H2O with photogenerated [RuIII(bipy)3]3+. Shortly before illumination (started at t = 0) the reaction mixture (H182O (5%), CaMn2O4 · H2O (1 mg ml−1), [Ru(bipy)3]2+ (1.5 mM), and [Co(NH3)5Cl]2+ (12.5 mM); pH ~4) inside the MIMS cell was purged with N2 until “zero” O2 level was reached. (C) Change in 18α-value for O2 production by PSII membrane fragments isolated from spinach. O2 evolution was induced by actinic continuous light at t = 0. Other conditions: 5% H182O, [Chl] = 0.03 mg ml−1, 0.6 mM PPBQ, 2 mM K3[Fe(CN)6], pH 6.0, and 20°C. Gray dashed lines in all panels indicates the theoretical 18α value expected for reaction of the “true” water-splitting, i.e., when both oxygen atoms of formed O2 originate from water. In all cases O2 production was detected by TR-MIMS as 16O2 (at m/z 32), 16O18O (m/z 34), and 18O2 (m/z 36), and the 18α was calculated according to the following equation: 18α = ([18O2] + 1/2[16O18O])/[O2]total. Adapted from Shevela et al. (2011b).
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