2004; Clausen et al. 2005). Related approaches can be taken to probe for example for binding sites of carbonate or hydrogencarbonate GSK3235025 in PSII (Shevela et al. 2008). In these experiments, it is attempted to replace the bound inorganic carbon (Ci) by the addition of a molecule (formate) that competes for the binding site, or by the destruction of the binding site via the addition of a strong
reductant. In both cases the released Ci is converted by the intrinsic or externally added CA into CO2 and can then be detected via the MIMS approach. 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 Selleck mTOR inhibitor al. 1991, 1997), while the destruction of the Mn4O x Ca cluster does not lead to a release of Ci. This demonstrates the absence of a tightly bound
Ci within the water oxidizing complex (see also Ulas et al. 2008; Aoyama et al. 2008). Fig. 6 Probing the binding of inorganic carbon (Ci) to photosystem II. The right side shows that the addition of formate to PSII induces a release of Ci into the medium which is clearly above the background measured by injection of formate into buffer. The released Ci is converted to CO2 by the intrinsic carbonic anhydrase (CA) activity of thylakoids and by added CA. The released CO2 corresponds to about 0.3 Ci/PSII. Left side: addition of hydroxylamine at concentrations known to rapidly reduce Carbohydrate the Mn4OxCa cluster and to release the manganese as Mn(II) into the medium did not lead to CO2 signals above background (left side). 15N-labeled hydroxylamine was used to shift the click here signal of N2O, which is produced during the reduction, to mass 46 Real time isotopic fractionation Isotopic fractionation is the ratio of one isotopic species (isotopologue) over another and brings with it information about chemical reactions. The fractionation can be due to (1) chemical diffusion such as CO2 assimilation in leaves (Farquhar et al. 1989), or to chemical
reactions where (2) there is a kinetic isotope effect (KIE, i.e., an isotope dependant difference in reaction rate) or (3) an equilibrium isotope effect (EIE, i.e., a change in the equilibrium concentration of an isotopic species). Traditionally measurements are typically performed with a time-dependent sampling of the concentrations of the products (e.g., Guy et al. 1993; Tian and Klinman 1993; Ribas-Carbo et al. 2005). This technique usually requires chromatographic separation or molecular sieve/freeze trapping of gases prior to analysis, and in the case of molecular oxygen, its initial conversion into CO2. Alternatively, such experiments can also be undertaken as real-time continuous measurement of gas concentrations using a MIMS approach. In this case, both reaction rates (i.e., given as ∆O2) and the absolute concentration of substrate (i.e., [O2]) are measured simultaneously for unlabeled and labeled isotopes.