We show that MALDI mass spectrometry, suitable for mixtures, is an indispensable tool in probing the mechanism of nanocluster synthesis enabling positive identification of nanoclusters. The size evolution of the mixture of larger clusters (Au(102), Au(68), Au(38)) to form highly monodisperse Au(25) nanoclusters is demonstrated and probably includes the participation of Au(I) thiolate. The size evolution via structural reconstruction of the larger cores such as 38, approximately 44, 68, and 102 to a Au(25) nanocluster has been discussed.

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...

, and ؉18 daltons. The masses of the modifications suggest that the tryptophan is modified to kynurenine (؉4), a keto-͞ amino-͞hydroxy-(؉16) derivative, and a dihydro-hydroxy-(؉18) derivative of the indole side chain. Peptide synthesis and MS͞MS confirmed the kynurenine assignment. The ؉16 and ؉18 tryptophan modifications may be intermediates formed during the oxidative cleavage of the indole ring to give kynurenine. The sitedirected mutations, W352C, W352L, and W352A, exhibit an increased rate of photoinhibition relative to wild type. We hypothesize that Trp-352 oxidative modifications are a byproduct of PSII water-splitting or electron transfer reactions and that these modifications target PSII for turnover. As a step toward understanding the tertiary structure of this CP43 peptide, structural modeling was performed by using molecular dynamics.mass spectrometry ͉ collision-induced dissociation ͉ tryptophan ͉ kynurenine ͉ photoinhibition P hotosystem II (PSII) is a protein-pigment complex located in thylakoid membranes of plants, eukaryotic algae, and cyanobacteria. PSII catalyzes the light-driven oxidation of water to O 2 , and the reduction of plastoquinone. PSII contains both intrinsic and extrinsic polypeptides. The intrinsic polypeptides include chlorophyll-binding proteins, CP47, CP43, and the D1 and D2 polypeptides (reviewed in ref. 1). The D2͞D1 heterodimer binds P 680 , pheophytin, and the quinone receptors, Q A and Q B (2). Three extrinsic subunits, the manganese stabilizing, 24-kDa, and 18-kDa proteins, are required for maximum oxygen evolution in plants (3, 4). Recently, a 3.8-Å structure of the cyanobacterial PSII reaction center has been reported (5, 6).The intrinsic PSII subunits, CP43 and CP47, function as light-harvesting proteins and play a role in PSII assembly and activity (7-11). CP47 and CP43 have similar tertiary and secondary structures (5). Each polypeptide has six membranespanning regions and a large luminal, hydrophilic loop (E) between helix V and VI (5, 7). In Synechocystis sp. PCC 6803, loop E of the CP43 subunit extends from residue Asn-280 to . Mutations or deletions in this loop inactivate or impair PSII activity in Synechocystis (9, 11-13).Posttranslational modifications can play important roles in the assembly, degradation, structure, and function of proteins. However, little is known about the roles of such modifications in membrane proteins. For example, in cytochrome c oxidase, a crosslinked tyrosine-histidine cofactor has been identified at the binuclear metal site (14); the function of this cofactor has not yet been definitively established. Recently, it has been suggested that posttranslationally modified amino acids, containing carbonyl groups, covalently bind hydrazines and amines at the catalytic site of PSII (10, 15). Because amines and hydrazines are inhibitors of photosynthetic water oxidation, it was suggested that these carbonyl-containing amino acids play roles in the structure, function, or assembly of PSII.To obtain more information about posttranslational mod...

The capsular polysaccharide of group B Streptococcus is a key virulence factor and an important target for protective immune responses. Until now, the nature of the attachment between the capsular polysaccharide and the bacterial cell has been poorly defined. We isolated insoluble cell wall fragments from lysates of type III group B Streptococcus and showed that the complexes contained both capsular polysaccharide and group B carbohydrate covalently bound to peptidoglycan. Treatment with the endo-N-acetylmuramidase mutanolysin released soluble complexes of capsular polysaccharide linked to group B carbohydrate by peptidoglycan fragments. Capsular polysaccharide could be enzymatically cleaved from group B carbohydrate by treatment of the soluble complexes with ␤-Nacetylglucosaminidase, which catalyzes hydrolysis of the ␤-D-GlcNAc(134)␤-D-MurNAc subunit produced by mutanolysin digestion of peptidoglycan. Evidence from gas chromatography/mass spectrometry and 31 P NMR analysis of the separated polysaccharides supports a model of the group B Streptococcus cell surface in which the group B carbohydrate and the capsular polysaccharide are independently linked to the glycan backbone of cell wall peptidoglycan; group B carbohydrate is linked to N-acetylmuramic acid, and capsular polysaccharide is linked via a phosphodiester bond and an oligosaccharide linker to N-acetylglucosamine.

Fatty acids, recently reported as constituents of certain fish lipids, were identified to be derivatives of furan (furanoid fish fatty acids). 12,15-Epoxy-13,14-dimethyleicosa-12,14-dienoic acid is predominant among the furan acids and is associated with bis-homologs in regard to chain length. Monomethyl acids, such as 12,15-epoxy-13-methyleicosa-12,14-dienoic, are present in appreciable amounts. The structures were concluded from oxidative degradations, from mass spectrometry of methyl esters of the novel acids and fatty acids derived from them by opening the ring, and from nuclear magnetic resonance, infrared, and Raman spectra. The results from chemical procedures and from spectrometric methods were in agreement with those obtained with authentic methyl 9,12-epoxyoctadeca-9,11-dienoate. The number of substituents at the furan ring greatly influences hydrogenation, hydrogenolysis, and hydrolysis reactions of the ring.

The role of surfactant (most often tetraoctylammonium bromide) in the two-phase Brust gold nanocluster synthesis (Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801) has been unclear. The results of the surfactant-free synthesis of gold nanoclusters in methylene chloride employing NaBH4 as a reducing agent with the usual synthetic conditions such as excess thiol:gold ratio (>3) and ice-cold reaction temperature are reported. A nanocluster mixture, ∼5% yield with Au(I)SR byproducts, was obtained. The isolated Au nanoclusters were characterized by mass spectrometry, UV−visible, and nuclear magnetic resonance spectroscopy. MALDI-TOF mass spectrometry reveals the presence of mixtures of nanoclusters in the Au16−Au31 size range. Discrete features in the UV−visible spectrum suggested the presence of small <2 nm particles. Relatively sharp peaks in NMR confirm the small size of the nanoclusters as suggested by MALDI-TOF MS. These results are in contrast to the commonly observed mixture of gold cores such as 25, 38, 102, and 144. It is proposed that the surfactant plays a major role in the gold nanocluster synthesis by its influence on the solubility of Au(I) species. The observed nanoclusters were analyzed in terms of the superatom complex model and found to contain 4 e−, 5 e−, 8 e−, and 10 e− nanoclusters.

Wild rice hulls (WRH) have not been utilized in any valuable manner. Minnesota WRH have been shown by us to possess antioxidant properties. The methanol extract of hulls showed antioxidant activity when added to ground beef, as evaluated by the content of thiobarbituric acid reactive substances (TBARS). The results of an ammonium thiocyanate assay also showed that some fractions of the hull methanol extract (MeOH:H 2 O, 75:25) have strong antioxidant activity. The yield of the evaporated methanol extract was 2.51% of WRH. The crude methanol extract was fractionated according to hydrophobicity. The antioxidant assay revealed that eluates of MeOH: H 2 O (50:50, 75:25) and absolute methanol have the strongest antioxidative activity in ground beef, as measured by the content of TBARS. Antioxidants were isolated from the 75:25 eluate and identified by mass spectrometry as 2,3,6-trimethylanisole (anisole); m-hydroxybenzaldehyde; 4-hydroxy-3-methoxybenzaldehyde (vanillin); and 4-hydroxy-3,5-dimethoxybenzaldehyde (syringaldehyde). Another compound identified, 2,3-dihydrobenzofuran, was a prooxidant.

Furan fatty acids (F acids) have been found in the livers and/or testes of 20 species, representing 9 families, of male freshwater fish. In 9 species they are major components of the lipids while in the remaining 11 species they occur to a much lesser extent. The F acids in some species reach a maximum concentation in the testes lipids, and minimum liver lipid concentration, at spawning. In all species in the testes, the F acids are confined almost exclusively to the triglyceride fraction while, in the liver lipids, they are found, in order of decreasing concentration, in the cholesteryl esters, the triglycerides, and the phospholipids. In the lipids of many individuals F6, 12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid, is the major fatty acid present. It is presumed that these acids perform some as yet unidentified metabolic function. Isolation technology and identification of F acids by a specific thin layer chromatographic spray reagent are discussed.