Reactions of the metallacrown ethers, [PdCl2{PPh2(CH2CH2O)(n)CH2CH2PPh2-P,P'}](m) (n = 3, 5), with PdCl2 or Pd(PhCN)(2)Cl-2 yield the new dimetallacrown ethers, [Pd2Cl2(mu-Cl)(2){PPh2(CH2CH2O)(n)CH2CH2PPh2- P, P'}](m) (n = 3 (3), n = 5 (4)). Similar reactions of [PdCl2{PPh2(CH2)(12)PPh2- P, P'}](m) and [PdCl2{PPh2(CH2CH2O)(2)CH2CH3-P}(2)] with PdCl2 or Pd(PhCN)(2)Cl-2 yield [Pd2Cl2(mu-Cl-2){PPh2(CH2)(12)PPh2-P,P'}](m) (5) and [Pd2Cl2(mu-Cl)(2){PPh2(CH2CH2O)(2)CH2CH3- P}(2)] ( 6), respectively. The chloride-bridged dimetallacrown ethers, 3 and 4, are cleanly converted into the iodide-bridged dimetallacrown ethers, [Pd2I2(mu-I)(2){PPh2(CH2CH2O)(n)CH2CH2PPh2- P, P'}](m) ( n = 3 ( 7), n = 5 ( 8)) by reaction with excess NaI. In solution, 3 and 4 exist as mixtures of syn monomers and cyclic oligomers while 7 and 8 exist as mixtures of both syn and anti monomers and cyclic oligomers. The solid state structures of syn-[Pd2I2(mu-I)(2){ PPh2(CH2CH2O)(3)CH2CH2PPh2- P, P'}] (syn-7) and of anti-[Pd2I2(mu-I)(2){Ph2P(CH2CH2O)(2)CH2CH3)(2)- P}(2)} (anti-9) have been determined. These structures are consistent with the major species present in the solutions. They also suggest that syn-7 experiences ring-strain, which is consistent with the results from the P-31{H-1} NMR studies.

Spontaneous formation of isoaspartyl residues (isoAsp) disrupts the structure and function of many normal proteins. Protein isoaspartyl methyltransferase (PIMT) reverts many isoAsp residues to aspartate as a protein repair process. We have determined the crystal structure of human protein isoaspartyl methyltransferase (HPIMT) complexed with adenosyl homocysteine (AdoHcy) to 1.6-A resolution. The core structure has a nucleotide binding domain motif, which is structurally homologous with the N-terminal domain of the bacterial Thermotoga maritima PIMT. Highly conserved residues in PIMTs among different phyla are placed at positions critical to AdoHcy binding and orienting the isoAsp residue substrate for methylation. The AdoHcy is completely enclosed within the HPIMT and a conformational change must occur to allow exchange with adenosyl methionine (AdoMet). An ordered sequential enzyme mechanism is supported because C-terminal residues involved with AdoHcy binding also form the isoAsp peptide binding site, and a change of conformation to allow AdoHcy to escape would preclude peptide binding. Modeling experiments indicated isoAsp groups observed in some known protein crystal structures could bind to the HPIMT active site.

The correlation between the relative intensity of water Raman band and crystal quality was studied for thaumatin and tetragonal lysozyme crystals grown under different conditions. The intensity variation of the band, revealed for the set of crystals was interpreted as being due to the different extents of ordering of internal water molecules. It was suggested that ordering is mainly relative to the alignment of the angle between the O–H bonds in water, since this angle serves as a normal coordinate for the symmetric bending vibration of H–O–H unit and the spectral width of the corresponding Raman band (2ν2) at 3212cm−1 appeared to be the main indicator of ordering. The assumption that the ordering of internal water molecules is relative to the overall protein crystal perfectness was verified by comparison of crystal scores obtained via the Raman and diffraction methods. The assessment of the crystal perfectness via these two methods seems to show some correlation. If this correlation confirmed, the noninvasive Raman spectroscopy may be used to monitor crystal quality during its growth.

Quantitative P-31{H-1} NMR spectroscopic studies of monomer-oligomer and cis-trans equilibria in chloroform-d solutions of PdCl2{Ph2P(CH2CH2O)(n)CH2CH2PPh2-P,P'}(n) n = 3, 4, 5) metallacrown ethers and of PdCl2{Ph2P(CH2)(12)PPh2-P,P'} are reported. The NMR data for all of the complexes can be adequately modeled using a single cis-trans isomerization equilibrium and two step-polymerization (dimerization, oligomerization) equilibria. As expected, both the dimerization and oligomerization equilibrium constants for the metallacrown ethers increase as n increases. However, the dimerization equilibrium constants of the metallacrown ethers are much smaller than are the oligomerization equilibrium constants. In contrast, the dimerization and oligomerization equilibrium constants for PdCl2{Ph2P(CH2)(12)PPh2-P,P'} are nearly identical and are significantly larger than are those of the metallacrown ethers. Kinetic studies of monomer-oligomer and cis-trans equilibria in solutions of cis-PdCl2{Ph2P(CH2CH2O)(3)CH2CH2PPh2-P,P'} indicate that the trans monomer and trans oligomers are formed at approximately the same rates and that the reactions follow reversible first-order kinetics. The very different dimerization constants for the metallacrown ethers and PdCl2{Ph2P(CH2)(12)PPh2-P,P'}and the similar rates of formation for the trans monomer and trans oligomers suggest that the isomerization and dimerization reactions have a common rate-determining step that involves cleavage of a palladium-phosphorus bond.

Peroxynitrite (ONOO−), the reaction product of superoxide (O2−) and nitric oxide (NO), may be a major cytotoxic agent produced during inflammation, sepsis, and ischemia/reperfusion. Bovine Cu,Zn superoxide dismutase reacted with peroxynitrite to form a stable yellow protein-bound adduct identified as nitrotyrosine. The uv-visible spectrum of the peroxynitrite-modified superoxide dismutase was highly pH dependent, exhibiting a peak at 438 nm at alkaline pH that shifts to 356 nm at acidic pH. An equivalent uv-visible spectrum was obtained by Cu,Zn superoxide dismutase treated with tetranitromethane. The Raman spectrum of authentic nitrotyrosine was contained in the spectrum of peroxynitrite-modified Cu,Zn superoxide dismutase. The reaction was specific for peroxynitrite because no significant amounts of nitrotyrosine were formed with nitric oxide (NO), nitrogen dioxide (NO2), nitrite (NO2−), or nitrate (NO3−). Removal of the copper from the Cu,Zn superoxide dismutase prevented formation of nitrotyrosine by peroxynitrite. The mechanism appears to involve peroxynitrite initially reacting with the active site copper to form an intermediate with the reactivity of nitronium ion (NO2+), which then nitrates tyrosine on a second molecule of superoxide dismutase. In the absence of exogenous phenolics, the rate of nitration of tyrosine followed second-order kinetics with respect to Cu,Zn superoxide dismutase concentration, proceeding at a rate of 1.0 ± 0.1 m−1 · s−1. Peroxynitrite-mediated nitration of tyrosine was also observed with the Mn and Fe superoxide dismutases as well as other copper-containing proteins.

The crystal structure of bovine Cu,Zn superoxide dismutase modified with peroxynitrite (ONOO-) was determined by X-ray diffraction, utilizing the existing three-dimensional model of the native structure deposited in the Brookhaven Protein Data Bank (J. A. Tainer et al., J. Mol. Biol. 160, 181-217, 1982). The native structure and the modified derivative were refined to R factors of 19.0 and 18.7% respectively using diffraction data from 6.0 to 2.5 A. The major result after reaction with peroxynitrite was the appearance of electron density 1.45 A from a single epsilon carbon of Tyr-108, the only tyrosine residue in the sequence. Tyr-108 is a solvent-exposed residue 18 A from the copper atom in the active site. The electron density was consistent with nitration of Tyr-108 at one of the epsilon carbons to form 3-nitrotyrosine. We propose that the nitration occurs in solution by transfer of a nitronium-like species from the active site on one superoxide dismutase dimer to the Tyr-108 of a second dimer.