The kinetics of water exchange across the chloroplast membrane

The kinetics of water exchange across the membrane of class II chloroplasts has been studied by two NMR methods. Both methods utilize Dy(en)3+ (en = ethylenediamine) to induce a transmembranal chemical shift the order of 40 Hz in the water proton resonance. The shift reagent is impermeant to the chloroplast membrane, inert as a redox reagent, soluble at millimolar concentrations at neutral pH, and associated with a large, virtually temperature independent molar shift (0.10-0.12 ppm/mM). Water exchange across the membrane is monitored by two independent experiments. In the first, chemical exchange causes line broadening in the water proton resonance in the high-resolution spectrum. Measurement of the incremental linewidth as a function of transmembranal chemical shift determines the exchange kinetics as well as the fractions of water protons in internal and external media. In the second experiment, chemical exchange causes the transverse relaxation time, as measured by the Carr-Purcell-Gill-Meiboom technique, to be dependent on the 180 degree pulse spacing. The two experiments, while independent of each other, depend on the same set of theoretical parameters. These parameters are overdetermined by simultaneous analysis of both experiments. The mean lifetime of a water proton in the inner thylakoid space is found to be 1.1 +/- 0.8 ms at 25 degrees C and 2.75 +/- 0.4 ms at 3 degrees C in NH2OH/EDTA-treated chloroplasts. Values derived from dark-adapted chloroplasts that are active with respect to oxygen evolution are 1.1 +/- 0.3 ms (25 degrees C) and 1.75 +/- 0.4 ms (3 degrees C). The internal thylakoid volume is also determined in principle by the data, but uncertainties in the membrane volume and the transmembranal chemical shift severely limits the accuracy of this measurement.