The thermostability of manganese stabilizing protein of photosystem II was examined by biochemical and spectroscopic techniques. Samples of both native and recombinant spinach manganese stabilizing protein incubated at 90 degreesC and then cooled to 25 degreesC were capable of rebinding to, and of reactivating, the O2-evolution activity of photosystem II membranes from which the native protein had been removed. Far-UV circular dichroism and FT-IR spectroscopies were used to analyze the structural consequences of heating manganese stabilizing protein. The data obtained from these techniques show that heating causes a complete loss of the protein's secondary structure, and that this is a reversible, noncooperative phenomenon. Upon cooling, the secondary structures of the heat-treated proteins return to a state similar to, but not identical with, that of the native, unheated controls. Restoration of a near-native tertiary structure is confirmed both by size-exclusion chromatography and by near-UV circular dichroism. The functional and structural thermostability of manganese stabilizing protein reported here, in conjunction with additional known properties of this protein (acidic pI, high random coil and turn content, anomalous hydrodynamic behavior), identifies manganese stabilizing protein as a natively unfolded protein [Weinreb et al. (1996) Biochemistry 35, 13709-13715]. Although these proteins lack amino acid sequence identity, their functional solution conformations under physiological conditions are said to be "natively unfolded". We suggest that, as with other members of this family of proteins, the natively unfolded structure of manganese stabilizing protein facilitates the highly effective protein-protein interactions that are necessary for its assembly into photosystem II.

Photosystem II catalyzes the light-driven oxidation of water and reduction of plastoquinone in oxygenic photosynthesis. The manganese stabilizing protein (MSP) of photosystem II is an extrinsic subunit that plays an important role in catalytic activity. This subunit can be extracted and re-bound to the photosystem II reaction center. Extraction is associated with decreased stability of manganese binding by the enzyme and by loss in high rates of oxygen evolution activity; reconstitution reverses these phenomena. Since little is known about the assembly of complex membrane proteins, we have employed isotope editing and vibrational spectroscopy to obtain information about any changes in secondary structure that occur in MSP upon functional reconstitution to photosystem II. The spectroscopic data obtained are consistent with substantial changes in conformation when MSP binds to photosystem II; approximately 30-40% of the peptide backbone undergoes a change in secondary structure. These conclusions were reached by comparing different aliquots, before and after binding, of the same 13[C]MSP sample. Analysis of amide I band line shapes through Fourier deconvolution and nonlinear regression suggests that binding of MSP to photosystem II is associated with a decrease in random structure and an increase in beta-sheet content. We conclude that binding of MSP to the reaction center can induce folding of MSP. Our results also indicate that, in solution, MSP can sample a variety of conformational states, which differ in hydrogen bonding of the peptide backbone.

Plastoquinone (PQ-9) is active as an electron/proton transfer component in photosynthetic membranes. For example, in the photosynthetic complex, photosystem II (PSII), PQ-9 acts as Q A , a one-electron acceptor, and as Q B , a two electron, two proton accepting species. Light-minus-dark difference Fourier transform infrared (FT-IR) spectroscopy is a technique with which mechanistic information can be obtained concerning PSII. Here, we present combined experimental and computational studies designed to identify the vibrational contributions of the electron acceptor, Q A , in its oxidized and one-electron reduced states to the difference FT-IR spectrum. Infrared spectra of decyl-PQ and PQ-9 were obtained; the difference infrared spectra associated with the formation of the corresponding anion radicals were also generated in ethanol solutions. Vibrational mode assignments were made based on hybrid Hartree-Fock/density functional (HF/DF) B3LYP calculations with a 6-31G(d) basis set. Calculations were performed for hydrogen bonded models of PQ-1 and its radical anion. In addition, a methionine-tolerant strain of the cyanobacterium, Synechocystis sp. PCC 6803, was used to deuterate PQ-9 in PSII. The macrocycle and phytol tail of chlorophyll were not labeled by this procedure. Mass spectral data may be consistent with partial 13 3 methoxy labeling of chlorophyll. Lack of phytol labeling implies that carotenoids were unlabeled. Difference FT-IR spectra were then obtained by illumination at 80 K, resulting in the one-electron reduction of Q A . When spectra were obtained of PSII preparations, in which 39% of PQ was 2 H 3 labeled and 48% was 2 H 6 labeled, isotope-induced shifts were observed. Comparison of these data to vibrational spectra obtained in vitro and to mode frequencies and intensities from B3LYP/ 6-31G(d) calculations provides the basis for vibrational mode assignments.Photosystem II (PSII), a membrane-associated pigmentprotein complex, carries out the oxidation of water and reduction of PQ-9 (plastoquinone-9) in all oxygen-evolving plants, algae, and cyanobacteria. Photoexcitation of the primary electron donor, P 680 , results in electron transfer to a bound PQ-9, called Q A , via a pheophytin molecule. Reduced Q A is reoxidized by an exchangeable PQ-9, named Q B . Q A functions as a oneelectron acceptor, and the reduced form, Q A -, is an unprotonated semiquinone anion radical. Q B , on the other hand, is a twoelectron, two-proton acceptor {reviewed in ref 1}. Electron transfer events on the acceptor side of PSII resemble reactions occurring on the acceptor side of the photosynthetic bacterial reaction center. 2 This enzyme, for which high-resolution structural information is available, uses UQ (ubiquinone) or menaquinone, instead of PQ-9, as acceptor molecules {reviewed in ref 3}.On the donor side of PSII, P 680 + is reduced by a redox-active tyrosine, Z. 4-8 The tyrosine radical, Z • , is reduced by a multinuclear manganese cluster on the microsecond to millisecond time range {see ref 9 and references ther...