Flash-Induced FTIR Difference Spectroscopy Shows No Evidence for the Structural Coupling of Bicarbonate to the Oxygen-Evolving Mn Cluster in Photosystem II
Abstract:Bicarbonate is known to be required for the maximum activity of photosystem II. Although it is well established that bicarbonate is bound to the nonheme iron to regulate the quinone reactions, the effect of bicarbonate on oxygen evolution is still controversial, and its binding site and exact physiological roles remain to be clarified. In this study, the structural coupling of bicarbonate to the oxygen-evolving center (OEC) was studied using Fourier transform infrared (FTIR) difference spectroscopy. Flash-indu… Show more
“…Figure 6 demonstrates that injection of formate releases carbonate/hydrogencarbonate from the non-heme iron at the acceptor side of PSII (see also Govindjee et al 1991, 1997), while the destruction of the Mn 4 O x Ca cluster does not lead to a release of C i . This demonstrates the absence of a tightly bound C i within the water oxidizing complex (see also Ulas et al 2008; Aoyama et al 2008). Fig.…”
Significant insights into plant photosynthesis and respiration have been achieved using membrane inlet mass spectrometry (MIMS) for the analysis of stable isotope distribution of gases. The MIMS approach is based on using a gas permeable membrane to enable the entry of gas molecules into the mass spectrometer source. This is a simple yet durable approach for the analysis of volatile gases, particularly atmospheric gases. The MIMS technique strongly lends itself to the study of reaction flux where isotopic labeling is employed to differentiate two competing processes; i.e., O2 evolution versus O2 uptake reactions from PSII or terminal oxidase/rubisco reactions. Such investigations have been used for in vitro studies of whole leaves and isolated cells. The MIMS approach is also able to follow rates of isotopic exchange, which is useful for obtaining chemical exchange rates. These types of measurements have been employed for oxygen ligand exchange in PSII and to discern reaction rates of the carbonic anhydrase reactions. Recent developments have also engaged MIMS for online isotopic fractionation and for the study of reactions in inorganic systems that are capable of water splitting or H2 generation. The simplicity of the sampling approach coupled to the high sensitivity of modern instrumentation is a reason for the growing applicability of this technique for a range of problems in plant photosynthesis and respiration. This review offers some insights into the sampling approaches and the experiments that have been conducted with MIMS.
“…Figure 6 demonstrates that injection of formate releases carbonate/hydrogencarbonate from the non-heme iron at the acceptor side of PSII (see also Govindjee et al 1991, 1997), while the destruction of the Mn 4 O x Ca cluster does not lead to a release of C i . This demonstrates the absence of a tightly bound C i within the water oxidizing complex (see also Ulas et al 2008; Aoyama et al 2008). Fig.…”
Significant insights into plant photosynthesis and respiration have been achieved using membrane inlet mass spectrometry (MIMS) for the analysis of stable isotope distribution of gases. The MIMS approach is based on using a gas permeable membrane to enable the entry of gas molecules into the mass spectrometer source. This is a simple yet durable approach for the analysis of volatile gases, particularly atmospheric gases. The MIMS technique strongly lends itself to the study of reaction flux where isotopic labeling is employed to differentiate two competing processes; i.e., O2 evolution versus O2 uptake reactions from PSII or terminal oxidase/rubisco reactions. Such investigations have been used for in vitro studies of whole leaves and isolated cells. The MIMS approach is also able to follow rates of isotopic exchange, which is useful for obtaining chemical exchange rates. These types of measurements have been employed for oxygen ligand exchange in PSII and to discern reaction rates of the carbonic anhydrase reactions. Recent developments have also engaged MIMS for online isotopic fractionation and for the study of reactions in inorganic systems that are capable of water splitting or H2 generation. The simplicity of the sampling approach coupled to the high sensitivity of modern instrumentation is a reason for the growing applicability of this technique for a range of problems in plant photosynthesis and respiration. This review offers some insights into the sampling approaches and the experiments that have been conducted with MIMS.
“…The sample in the cell was separated from the vacuum (3 × 10 −8 bar) of the mass spectrometer via a 1-cm-diameter inlet that was covered by a 25-μm-thick silicon membrane (MEM-213; Mempro) resting on a porous Teflon support (Ø 10 mm). In the mass spectrometer the gases were ionized by electron impact and separated by a magnetic sector field into a seven-cup Faraday detector array for simultaneous detection of 16 (m/z 48). The simultaneous recording of argon (m/z 40) served as a control.…”
Section: Methodsmentioning
confidence: 99%
“…11 and 12). Although a tight binding of C i near the Mn 4 CaO 5 cluster is excluded on the basis of X-ray crystallography (3,14), FTIR spectroscopy (16), and mass spectrometry (17,18), the possibility that a weakly bound HCO − 3 affects the activity of PSII at the donor side remains a viable option (reviewed in refs. 10 and 19).…”
Cyanobacteria, algae, and plants oxidize water to the O 2 we breathe, and consume CO 2 during the synthesis of biomass. Although these vital processes are functionally and structurally well separated in photosynthetic organisms, there is a long-debated role for CO 2 /HCO − 3 in water oxidation. Using membrane-inlet mass spectrometry we demonstrate that HCO − 3 acts as a mobile proton acceptor that helps to transport the protons produced inside of photosystem II by water oxidation out into the chloroplast's lumen, resulting in a light-driven production of O 2 and CO 2 . Depletion of HCO − 3 from the media leads, in the absence of added buffers, to a reversible down-regulation of O 2 production by about 20%. These findings add a previously unidentified component to the regulatory network of oxygenic photosynthesis and conclude the more than 50-y-long quest for the function of CO 2 / HCO − 3 in photosynthetic water oxidation.O xygenic photosynthesis in cyanobacteria, algae, and higher plants leads to the reduction of atmospheric CO 2 to energy-rich carbohydrates. The electrons needed for this process are extracted in a cyclic, light-driven process from water that is split into dioxygen (O 2 ) and protons. This reaction is catalyzed by a penta-μ-oxo bridged tetra-manganese calcium cluster (Mn 4 CaO 5 ) within the oxygen-evolving complex (OEC) of photosystem II (PSII) (1-4). The possible roles of inorganic carbon, C i ðC i = CO 2 ; H 2 CO 3 ; HCO − 3 ; CO 2− 3 Þ, in this process have been a controversial issue ever since Otto Warburg and Günter Krippahl (5) reported in 1958 that oxygen evolution by PSII strictly depends on CO 2 and therefore has to be based on the photolysis of H 2 CO 3 ("Kohlensäure") and not of water. These first experiments were indirect and, as became apparent later, were wrongly interpreted (6-8). Several research groups followed up on these initial results and identified two possible sites of C i interaction within PSII (reviewed in refs. 9-12). Functional and spectroscopic studies showed that HCO − 3 facilitates the reduction of the secondary plastoquinone electron acceptor (Q B ) of PSII by participating in the protonation of Q 2− B . Binding of HCO − 3 (or CO 2− 3 ) to the nonheme Fe between the quinones Q A and Q B was recently confirmed by X-ray crystallography (3,13,14). Despite this functional role at the acceptor side, the very tight binding of HCO − 3 to this site makes it impossible for the activity of PSII to be affected by changing the C i level of the medium; instead inhibitors such as formate need to be added to induce the acceptor-side effect (15). Consequently, the watersplitting electron-donor side of PSII has also been studied intensively (for recent reviews, see refs. 11 and 12). Although a tight binding of C i near the Mn 4 CaO 5 cluster is excluded on the basis of X-ray crystallography (3, 14), FTIR spectroscopy (16), and mass spectrometry (17, 18), the possibility that a weakly bound HCO − 3 affects the activity of PSII at the donor side remains a viable option (reviewed i...
“…Both approaches clearly demonstrated that the detected CO 2 /HCO − 3
does not originate from the inorganic core of the water-splitting site of PSII (Shevela et al, 2008a,b). Independent evidence for absence of HCO − 3 bound to the WOC was provided by FTIR and GS-MS experiments (Aoyama et al, 2008; Ulas et al, 2008). Moreover, in recent x-ray crystallography studies at resolutions of 1.9-3.0 Å no HCO − 3 was found in the vicinity of the Mn 4 CaO 5 cluster, while they all clearly show HCO − 3 bound to the NHI on the acceptor side of PSII (Guskov et al, 2010; Umena et al, 2011) (also, see Figure 1).…”
Section: Is Hydrogen Carbonate a Ligand To The Woc?mentioning
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 18O-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.
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