The hammerhead ribozyme is a catalytic RNA that requires divalent metal cations for activity under moderate ionic strength. Two important sites that are proposed to bind metal ions in the hammerhead ribozyme are the A9/G10.1 site, located at the junction between stem II and the conserved core, and the scissile phosphate (P1.1). (31)P NMR spectroscopy in conjunction with phosphorothioate substitutions is used in this study to investigate these putative metal sites. The (31)P NMR feature of a phosphorothioate appears in a unique spectral window and can be monitored for changes upon addition of metals. Addition of 1-2 equiv of Cd(2+) to the hammerhead with an A9-S(Rp) or A9-S(S)(Rp) substitution results in a 2-3 ppm upfield shift of the (31)P NMR resonance. In contrast, the P1.1-S(Rp) and P1.1-S(Sp) (31)P NMR features shift slightly and in opposite directions, with a total change in delta of

, 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...

A metal site in a 5'-GAAA-3' tetraloop, a stabilizing and phylogenetically conserved RNA motif, is explored using (31)P NMR spectroscopy and phosphorothioate modifications. Similar to previous reports [Legault, P., and Pardi, A. (1994) J. Magn. Reson., Ser. B 103, 82-86], the (31)P NMR spectrum of a 12-nucleotide stem-loop sequence 5'-GGCCGAAAGGCC-3' exhibits resolved features from each of the phosphodiester linkages. Titration with Mg(2+) results in distinct shifts of a subset of these (31)P features, which are assigned to phosphodiesters 5' to A6, A7, and G5. Titration with Co(NH(3))(6)(3+) causes only a slight upfield shift in the A6 feature, suggesting that changes caused by Mg(2+) are due to inner-sphere metal-phosphate coordination. R(p)-Phosphorothioate substitutions introduced enzymatically 5' to each of the three A residues of the tetraloop provide well-resolved (31)P NMR features that are observed to shift in the presence of Cd(2+) but not Mg(2+), again consistent with a metal-phosphate site. Analysis of (31)P NMR spectra using the sequence 5'-GGGCGAAAGUCC-3' with single phosphorothioate substitutions in the loop region, separated into R(p) and S(p) diastereomers, provides evidence for an inner-sphere interaction with the phosphate 5' to A7 but outer-sphere or structural effects that cause perturbations 5' to A6. Introduction of an R(p)-phosphorothioate 5' to A7 results in a distinct (31)P NMR spectrum, consistent with thermodynamic studies reported in the accompanying paper that indicate a unique structure caused by this substitution. On the basis of these results and existing structural information, a metal site in the 5'-GAAA-3' tetraloop is modeled using restrained molecular dynamics simulations.