ATP-binding cassette (ABC) transport proteins catalyze the translocation of substrates at the expense of hydrolysis of ATP, but the actual ATP/substrate stoichiometry is still controversial. In the osmoregulated ABC transporter (OpuA) from Lactococcus lactis, ATP hydrolysis and substrate translocation are tightly coupled, and the activity of right-side-in and inside-out reconstituted OpuA can be determined accurately. Although the ATP/substrate stoichiometry determined from the uptake of glycine betaine and intravesicular ATP hydrolysis tends to increase with decreasing average size of the liposomes, the data from inside-out reconstituted OpuA indicate that the mechanistic stoichiometry is 2. Moreover, the two orientations of OpuA in proteoliposomes allowed possible contributions from substrate (glycine betaine) inhibition on the trans-side of the membrane and inhibition by ADP to be determined. Here we show that OpuA is not inhibited by up to 400 mM glycine betaine on the trans-side of the membrane. ADP is an inhibitor, but accumulation of ADP was negligible in the assays with inside-out-oriented OpuA, and potential effects of the ATP/ADP ratio on the ATP/substrate stoichiometry determinations could be eliminated. ATP-binding cassette (ABC)1 transporters, found in both prokaryotes and eukaryotes, play an important role in various physiological processes ranging from uptake of nutrients, multidrug resistance, secretion of signal molecules or toxins, cell volume regulation, and other processes (reviewed in Refs. 1-4). Despite the diversity of functions and substrates of the ABC transporters, all members fuel the translocation process by ATP hydrolysis. Also, the basic architecture of ABC transporters is remarkably similar among members in the superfamily. Always present are two cytoplasmic-exposed nucleotide-binding domains, the ATP-binding cassettes, and two hydrophobic domains that are predicted to span the membrane multiple times in an ␣-helical conformation (2). The conserved assembly and arrangement of these domains have been clearly demonstrated in the recently determined structure of BtuCD, the ABC transporter that mediates uptake of vitamin B12 in Escherichia coli (5). Prokaryotic ABC transporters involved in solute uptake use an additional substrate-binding protein that delivers the substrate to the translocator. The ABC transporter, OpuA, from Lactococcus lactis belongs to a subfamily of the OTCN family of which members have the substrate-binding domain fused to the translocator (6). In the dimeric complex and presumably functional state, two of these chimeric substrate-binding/translocator proteins and two ATPase subunits are present. Extensive characterization of OpuA has revealed that this protein responds to osmotic stress, which is sensed at the cytoplasmic face as a change in ionic strength (7-9).The issue of ATP/substrate stoichiometry for members of the ABC transport family is still a subject of debate. ATP/substrate stoichiometries for a variety of ABC transporters range from 1 to 50 (10 -20). ...

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...

Photosystem II, the photosynthetic water-oxidizing complex, can be isolated from both plants and cyanobacteria. A variety of methods have been developed for purification of this enzyme, which can be isolated in several functional and structural forms. Knowledge of the pigment content of photosystem II preparations is important for precise spectroscopic, biochemical, and functional analysis. We have determined pigment stoichiometries in oxygen-evolving photosystem II preparations from plants and cyanobacteria. We have employed a solvent system for the isocratic elution of a reverse phase HPLC column in which we have determined the extinction coefficients of the relevant pigments. Pigments were extracted from four photosystem II preparations. These preparations included spinach photosystem II membranes [Berthold, D. A., Babcock, G. T., & Yocum, C. F. (1981) FEBS Lett. 134, 231-234], spinach photosystem II reaction center complexes [Ghanotakis, D. F., & Yocum, C. F. (1986) FEBS Lett. 197, 244-248], spinach photosystem II complexes [MacDonald, G. M., & Barry, B. A. (1992) Biochemistry 31, 9848-9856], and photosystem II particles isolated from the cyanobacterium, Synechocystis sp. PCC 6803 [Noren, G. H., Boerner, R. J., & Barry, B. A. (1991) Biochemistry 30, 3943-3950]. Pigment stoichiometries were determined using two different methods of data analysis and were based on the assumption that there are two pheophytin a molecules per photosystem II reaction center. The pigment stoichiometries obtained were comparable for the two methods of data analysis and agreed with previous biophysical and biochemical characterizations of the preparations. The average pigment stoichiometries (chlorophyll:plastoquinone-9 per 2 pheophytin a) determined using the two data analysis methods were as follows: photosystem II membranes, 274:3.2; photosystem II reaction center complexes, 78:2.5; Synechocystis PS II particles, 55:2.4; photosystem II complexes, 121:2.0.

The lactose permease, encoded by the lacY gene of Escherichia coli, is an integral membrane protein that functions as a proton and lactose symporter. In this study, we have characterized a novel monodisperse, purified preparation of lactose permease, as well as functionally reconstituted lactose permease, using spectroscopic techniques. The purification of monodisperse lactose permease has been aided by the development of a lacY gene product containing an amino-terminal six histidine affinity tag. In the novel purification method described here, lactose permease is purified from beta-dodecyl maltoside-solubilized membrane vesicles using three sequential column steps: hydroxyapatite, nickel-nitriloacetic acid (Ni-NTA) affinity, and cation-exchange chromatography. The hydroxyapatite step was shown to be essential in reducing aggregation of the final purified protein. Amino acid composition analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis support the conclusion that the protein has been purified to greater than 90% homogeneity. The protein has been successfully reconstituted and has been shown to be active for lactose transport. Fourier transform infrared (FT-IR) spectroscopy has been performed on monodisperse lactose permease and on proteoliposomes containing functional lactose permease. FT-IR spectroscopy supports the conclusion that the monodisperse lactose permease preparation is 80% alpha-helical and stably folded at 20 degreesC; thermal denaturation is first detected at 70 degreesC. Because the purified protein is also readily susceptible to 2H exchange, these results suggest that the protein is conformationally flexible and that 2H exchange is facilitated as the result of conformational fluctuations from the folded state.