Difference FT-IR study of a novel biochemical preparation of photosystem II

Abstract

There are two redox-active tyrosines in photosystem II, the water-splitting complex, that form neutral tyrosine radicals. One of these tyrosine radicals, D., is stable and has an unknown function. The other redox-active tyrosine, Z, acts to transfer oxidizing equivalents from the primary chlorophyll donor of photosystem II to the manganese cluster, where water oxidation occurs. In an attempt to obtain more information about Z and its interaction with its environment, we have begun a study using Fourier-transform infrared (FT-IR) vibrational spectroscopy. To facilitate these studies, we have developed a procedure to isolate spinach photosystem II complexes with an antenna size of approximately 100-110 chlorophylls per reaction center. These complexes show an approximately 2-fold increase in the specific activity of oxygen evolution over the activity of the starting material, photosystem II membranes. Although fully solubilized in detergent, these complexes retain the 24- and 18-kDa extrinsic proteins and exhibit no calcium chloride requirement for optimal oxygen evolution. In manganese-depleted photosystem II samples, Z. can be accumulated in the light. In the dark, the tyrosine radical is reduced and reprotonated to form the neutral tyrosine. Since this process is reversible and light-dependent, we have used light-minus-dark difference FT-IR spectroscopy to observe the vibrational difference spectrum that is associated with the oxidation of this residue. As a control, EPR spectra were measured under identical conditions to assess the amount of Z. that accumulated in the light. We also hope to use difference FT-IR to identify the amino acid with which Z may form a hydrogen bond.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific Isotopic Labeling and Photooxidation-linked Structural Changes in the Manganese-stabilizing Subunit of Photosystem II

“…These PSII membranes were used directly for FT-IR spectroscopy at 277 K and were in a sucrose buffer containing 0.4 M sucrose, 50 mM MES-NaOH, pH 6.0, 60 mM NaCl, and 20 mM CaCl 2 . PSII complexes, for FT-IR spectroscopy at 200 K, were purified from the membranes via detergent solubilization and ion-exchange chromatography (45). The final buffer contained 25% glycerol, 50 mM MES-NaOH, pH 6.0, 0.05% lauryl maltoside, 7.5 mM CaCl 2 , and 0.26 M NaCl.…”

Deprotonation of the 33-kDa, Extrinsic, Manganese-stabilizing Subunit Accompanies Photooxidation of Manganese in Photosystem II

“…2). PSII complexes were purified from this material through the use of ion exchange chromatography (29) and were concentrated for FT-IR studies. This procedure, carried out in the presence of excess detergent, ensures that 12 C and 13 C MSP are specifically bound.…”

“…For some experiments, PSII complexes were purified directly from PSII membranes (29). Rates of oxygen evolution in these preparations were similar to the rates given above.…”

The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

“…In the case of membrane proteins and photoactive proteins, a variety of systems have been investigated using the approaches described in this review and first introduced for bacteriorhodopsin. A few examples are the photosystem II [116,152], photoactive yellow protein (PYP) [59,118,124,246]; Ca 2+ -ATPase [21,22,52,98,133]; green fluorescent protein (GFP) and potassium channels [92,230]. One measure of the growth of FTIR difference spectroscopy of biomolecules is shown in Fig.…”