Letter pubs.acs.org/JPCL
Calcium and the Hydrogen-Bonded Water Network in the Photosynthetic Oxygen-Evolving Complex Brandon C. Polander and Bridgette A. Barry* Department of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia 30332, United States S Supporting Information *
ABSTRACT: In photosynthesis, photosystem II evolves oxygen from water at a Mn4CaO5 cluster (OEC). Calcium is required for biological oxygen evolution. In the OEC, a water network, extending from the calcium to four peptide carbonyl groups, has recently been predicted by a high-resolution crystal structure. Here, we use carbonyl vibrational frequencies as reporters of electrostatic changes to test the presence of this water network. A single flash, oxidizing Mn(III) to Mn(IV) (the S1 to S2 transition), upshifted the frequencies of peptide CO bands. The spectral change was attributable to a decrease in CO hydrogen bonding. Strontium, which supports a lower level of steady state activity, also led to an oxidation-induced shift in CO frequencies, but treatment with barium and magnesium, which do not support activity, did not. This work provides evidence that calcium maintains an electrostatically responsive water network in the OEC and shows that OEC peptide carbonyl groups can be used as solvatochromic markers. SECTION: Biophysical Chemistry and Biomolecules
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and assigns positions to bound water molecules. At the OEC, two water molecules are predicted to bind manganese, and two are predicted to bind Ca (Figure 1C). In this structure, water is integrated into an extensive hydrogen bond network around the OEC, which includes peptide carbonyl groups, one or more Mn ions, and the OEC Ca ion. It should be noted that it is not known which S state is represented in the X-ray structure, and the number and positions of water molecules in the network may be responsive to S state advancement.20,21 However, this high-resolution X-ray structure2 provides an excellent starting place for investigation of water dynamics in the OEC. Recently, we used amide carbonyl vibrational frequencies to probe the intercalation of ammonia into the OEC network.22 It was concluded that ammonia displaces water in the network; the effect was reversed by the addition of trehalose. Transient EPR studies of YZ• decay were also consistent with an effect of ammonia on a hydrogen-bonded network in the OEC.23,24 These studies suggested that peptide carbonyl frequencies can be used as solvatochromic markers for dynamics in the water network. There are few existing methods to measure the dynamic role of water in biological systems, but infrared spectroscopy provides such a technique.25,26 FT-IR spectroscopy has previously been used to monitor solvent and protein relaxation events occurring after S state transitions on the seconds time scale.27−29 In particular, our group and others,30−33 have used FT-IR spectroscopy previously to
n plants, cyanobacteria, and algae, photosynthesis generates molecular oxygen from water. This process is essential to the maintenance of aerobic life on earth and is thus one of the most important processes in biology, yet the chemistry of O−O bond formation is still not understood. Photosystem II (PSII) catalyzes this light-driven oxidation of water and the accompanying reduction of plastoquinone (reviewed in1). PSII is a multisubunit enzyme complex that consists of 17 membrane-spanning domains and three extrinsic domains. The core hydrophobic subunits D1, D2, CP43, and CP47, bind the redox-active cofactors.2 The oxygen-evolving complex (OEC), which contains a Mn4CaO5 cluster (Figure 1A), is the site of water oxidation. Ca is essential for this activity,3−7 yet the mechanistic role of Ca remains enigmatic.8 Four sequential photo-oxidations of the OEC result in the release of molecular oxygen. Therefore, oxygen release fluctuates with period four. The sequentially oxidized states of the OEC are termed the Sn states (Figure 1B).9 A redox active tyrosine, YZ, acts as an electron transfer intermediary on each S state transition.10,11 S1 is the dark stable state in which one oxidizing equivalent is stored at the OEC. A single flash given to a dark-adapted PSII sample generates the S2 state (Figure 1B, boxed), which is associated with the oxidation of Mn(III) to Mn(IV).12 Oxygen is released on the transition from the S3 to S0 state.9 In Ca depleted (CD) preparations, the oxidation of Mn has been reported to occur in some studies,13−16 but see refs 17−19. See a more detailed discussion of this point in the Supporting Information. Later S state transitions are blocked in CD PSII (reviewed in ref 8). The most recent PSII crystal structure has been resolved at 1.9 Å resolution.2 This structure identifies ligands to the OEC © 2013 American Chemical Society
Received: January 10, 2013 Accepted: February 13, 2013 Published: February 19, 2013 786
dx.doi.org/10.1021/jz400071k | J. Phys. Chem. Lett. 2013, 4, 786−791
The Journal of Physical Chemistry Letters
Letter
Examination of the PSII structure (Figure 1A) suggests that at least one Ca-bound water molecule participates in the extensive hydrogen bond network around the OEC. Theoretical models for oxygen evolution predict that Ca is important in O−O bond formation. Some mechanisms propose that O−O bond formation occurs between Mn-bridging oxygens (see ref 35 and references therein), whereas others suggest that oxygen formation results after nucleophilic attack of a Ca-bound water molecule on a Mn-bound water molecule.1,36 In this work, we report new information concerning the role of Ca and the presence of an extended water network. We use peptide carbonyl frequencies and a divalent ion substitution protocol. PSII preparations were isolated from market spinach using Triton X-100 and octylthioglucoside (OTG).37,38 Typical steady state oxygen rates were 1600 μmol O2 (mg chl-hr)−1 at pH 6.0 and 1200 μmol O2 (mg chl-hr)−1 at pH 7.5. This preparation retains extrinsic PsbP and PsbQ subunits as isolated, and thus has a high binding affinity for Ca.39 Many procedures for Ca removal, previously described, involve a 1−2 M NaCl wash in the light, which removes the PsbP and PsbQ subunits and decreases the binding affinity for Ca.3,4,40 However, at high pH in OTG PSII, the PsbP and PsbQ subunits are lost spontaneously.22 Therefore, at high pH, endogenous Ca can be removed, without 2 M NaCl treatment, by treatment with ethylene glycol-bis(2-aminoethylether)N,N,N′,N′-tetraacetic acid (EGTA). EGTA selectively removes Ca, leaving Mn ions bound. These EGTA-treated samples (referred to as Ca depleted or CD-OEC) were inactive in oxygen evolution (
