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Due to the high viscosity and bulk complexity of crude oil samples, it poses a grand challenge to determine hydrogen sulfide (H2S) in them. Although many methods have been developed for the analysis of H2S in complex systems, these methods generally have complex processes, large analysis error and low sensitivity. In the study, based on the reaction between triazine and H2S by generating thiadiazine, a small volume of complex crude oil sample was introduced directly into a triazine solution using acetonitrile as solvent for transferring H2S into detectable species by organic mass spectrometry. The experimental parameters of solution composition, acetic acid content, reaction time and reaction temperature were optimized, and the method of paper spray ionization-mass spectrometry (PSI-MS) was established for rapid analysis of H2S in complex crude oil samples. An optimal performance was achieved when the reaction of triazine and H2S was carried out at 30 ℃ for 2 h with acetonitrile containing 0.1% acetic acid as solvent. The standard curve for the quantitative analysis of H2S was constructed according to the linear relationship between MS signal intensity of resulting thiadiazine and the concentration of H2S, which was applied for actual crude oil samples from different sources. The results showed that the linear range and the limit of detection (LOD) were 0.1-1 000 μg/L and 0.041 μg/L with intra-day precision of 3.04% and inter-day precision of 3.18%, respectively. The PSI-MS was also employed to determine H2S in different actual crude oil samples, and the spiked recovery was 91.7%-103.5% with relative standard deviation (RSD) of 1.2%-4.5%. This method has the advantages of easy operation, low cost, high sensitivity and less contamination to mass spectrometer, which is promising for the direct and fast analysis of H2S in complex crude oil samples.

Research has been conducted on the L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea. The X-ray crystal structures of this synthase has been examined and the high-resolution structures of the enzyme complexed with ligands are presented.

Site-directed mutagenesis of the active-site residues and substrate substitution studies point out to the importance of hydroxyl group for substrate recognition and catalysis by scytalone dehydratase.

The fungal enzyme scytalone dehydratase catalyzes a syn elimination on scytalone by protonating the carbonyl to form an enol. Researches are creating inhibitors of this enzyme to control rice blast.

Experiments were performed to investigate the hydride transfer between formate and nicotinamide adenine dinucleotide. The data showed the structure of the pyridine ring during the transition state after high level ab initio molecular orbital analyses were performed. The findings suggest the maintenance of the general structure of the transition state even under various environments as proven by the kinetic effects of the reaction in the gas phase and the isotope effects in model systems.

Molecular dynamics simulations of ground and transition states were made for the SN2 displacement of chlorine from 1,2-dichloroethane at the active site of the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. By comparing the molecular dynamics simulations for the enzyme-substrate complex and the enzyme transition-state structure, changes in th active site structure was observed. Likewise, hydrogen bonding changes were seen upon comparing near attack conformation structures to transition-state structures.

The ab initio structures of 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), semiquinone (PQQH), and dihydroquinone (PQQH2) have been determined and compared with ab initio structures of the (PQQ) [Ca.sup.2+], (PQQH)[Ca.sup.2+], and (PQQH2) [Ca.sup.2+] complexes as well as the x-ray structure of (PQQ)[Ca.sup.2+] bound at the active site of the methanol dehydrogenase (MDH) of methyltropic bacteria. Plausible mechanisms for the MDH oxidation of methanol involving the (PQQ)[Ca.sup.2+] complex are explored via ab initio computations and discussed. Considering the reaction of methanol with PQQ in the absence of [Ca.sup.2+], nucleophilic addition of methanol to the PQQ C-5 carbonyl followed by a retro-ene elimination is deemed unlikely due to large energy barrier. A much more favorable disposition of the methanol C-5 adduct to provide formaldehyde involves proton ionization of the intermediate followed by elimination of methoxide concerted with hydride transfer to the oxygen of the C-4 carbonyl. Much the same transition state is reached if one searches for the transition state beginning with Asp-303-[Mathematical Expression Omitted] general-base removal of the methanol proton of the (PQQ)[Ca.sup.2+] O(H)[CH.sub.3] complex concerted with hydride transfer to the oxygen at C-4. For such a mechanism the role of the [Ca.sup.2+] moiety would be to (i) contribute to the formation of the ES complex (ii) provide a modest decrease in the [pK.sub.a] of methanol substrate,; and (iii) polarize the oxygen at C-5.

Ab initio and semiempirical quantum mechanical methods have been used to explain the reaction mechanism of the nonenzymatic transmethylation of catechol by S-adenosylmethionine. The fast gas phase reaction involves no overall barrier, and the reaction profile is similar to a typical gas phase SN2 reaction. There is good agreement between determined kinetic isotope effects in the model reaction and the measured effects for the enzymatic reaction, indicating a state similar to a nonenzymatic reaction.

The semiempirical PM3 method, calibrated against ab initio HF/6-31+G(d) theory, has been used to elucidate the reaction of 1,2-dichloroethane (DCE) with the carboxylate of Asp-124 at the active site of haloalkane dehalogenase of Xanthobacter autothropicus. Asp-124 and 13 other amino acid side chains that make up the active site cavity (Glu-56, Trp-125, Phe-128, Phe-172, Trp-175, Leu-179, Val-219, Phe-222, Pro-223, Val-226, Leu-262, Leu-263, and His-289) were included in the calculations. The three most significant observations of the present study are that: (i) the DCE substrate and Asp-124 carboxylate, in the reactive ES complex, are present as an ion-molecule complex with a structure similar to that seen in the gas-phase reaction of Ac[O.sup.-] with DCE; (ii) the structures of the transition states in the gas-phase and enzymatic reaction are much the same where the structure formed at the active site is somewhat exploded; and (iii) the enthalpies in going from ground states to transition states in the enzymatic and gas-phase reactions differ by only a couple kcal/mol. The dehalogenase derives its catalytic power from: (i) bringing the electrophile and nucleophile together in a low-dielectric environment in an orientation that allows the reaction to occur without much structural reorganization; (ii) desolvation; and (iii) stabilizing the leaving chloride anion by Trp-125 and Trp-175 through hydrogen bonding.

The [S.sub.N]2 displacements of chloride ion from C[H.sub.3]Cl, [C.sub.2][H.sub.5]Cl, and [C.sub.2][H.sub.4][Cl.sub.2] by acetate and hydroxide ions have been investigated, using ab initio molecular orbital theory at the HF/6-31+G(d), MP2/6-31+G(d), and MP4/6-31+G(d) levels of theory. The central barriers (calculated from the initial ion-molecule complex) of the reactions, the differences of the overall reaction energies, and the geometries of the transition states are compared. Essential stereochemical changes before and after the displacement reactions are described for selected cases. The gas phase reactions of hydroxide with C[H.sub.3]Cl, [C.sub.2][H.sub.5]Cl, and [C.sub.2][H.sub.4][Cl.sub.2] have no overall barrier, but there is a small overall barrier for the reactions of acetate with C[H.sub.3]Cl, [C.sub.2][H.sub.5]Cl, and [C.sub.2][H.sub.4][Cl.sub.2]. A self-consistent reaction field solvation model was used to examine the [S.sub.N]2 reactions between methyl chloride and hydroxide ion and between 1,2-dichloroethane and acetate in solution. As expected, the reactions in polar solvent have a large barrier. However, the transition state structures determined by ab initio calculations change only slightly in the presence of a highly polar solvent as compared with the gas phase. We also calibrated the PM3 method for future study of an enzymatic [S.sub.N]2 displacement of halogen.

The uncatalyzed dehalogenation of 4-chlorobenzoyl CoA using appropriate model compounds was theoretically investigated. Factors foreseen to modulate the catalytic power of 4-chlorobenzoyl CoA dehalogenase were studied. The hydrolysis of the aryl-enzyme intermediate and a related nucleophilic aromatic substitition reaction between tetrachlorohydroquinone and glutathione was also investigated.

Ab initio molecular orbital calculations were performed using a continuum solvent model to examine the reaction pathway of glutathione S-transferase-catalyzed nucleophilic aromatic substitution of 1-chloro-2,4-dinitrobenzene in aqueous media and in the gas phase. The results for reactions in aqueous media, which were consistent with experimental results, showed a large free energy barrier and predicted the rate-determining step to be C-S bond formation.

Ab initio molecular orbital simulations of a salt-bridge were performed in three solvents and in the gas phase. The simulations, which were combined with reaction field theory, examined the role of solvent environment on the interaction between the guanidinium group and the carboxylic group of an arginine modeled by formate and guanidium ions. Specifically, the energy difference and the interconversion barrier between the zwitterionic complex and its neutral hydrogen-bonded form was quantified.

The structure of flavin in various oxidation and protonation states using lumiflavin as a model compound was examined with ab initio molecular orbital theory. Notwithstanding its protonation state or whether it participates in hydrogen bonding, oxidized flavin is always planar. Flavin semiquinone radicals' structures are planar or very close to planar, while the reduced flavin H3Fl(sub red) is bent, with a ring puckering angle of 27.3 degrees along the N5 and N10 axis.

The enzyme subtilisin Carlsberg is subjected to a 745-ps molecular dynamics simulation in a box of dimethyl sulfur oxide. The aqueous X-ray crystal structure serves as the basis for the starting coordinates of the enzyme and the crystallographic waters. Results indicate that the proteins structure is still not far away from the aqueous crystal structure even after 745 ps of simulation. However, some structural perturbations are observed in the enzyme.

The catalytic mechanism of human carbonic anhydrase II (HCAII) was studied by using a combined high-level ab initio and free energy perturbation approach. It was found that the mechanism involves hydration of CO2 and dehydration of bicarbonate. The bicarbonate proton in the X-ray structure of the HCAII-bicarbonate complex is hydrogen bound to Thr-199, while one of the bicarbonate oxygens is hydrogen bound to the main-chain NH of Thr-199. The study shows the molecular-level details of the mechanism for the mode of action of HCAII.

Research has been conducted on 2,2-bis(trifluoromethyl)-1,1-dicyanoethylene. The authors describe the reactions of this compound with 6,6-dimethylfulvene, and report that this reaction leads to Diels-Alder cycloadduct which is unstable and exists in equilibrium with the starting materials in less polar solvents.

The possible intermediates required in the oxidation of vitamin K were studied theoretically. An alternative pathway is proposed which better explains empirical findings. There are two possible pathways for the formation of vitamin K 2,3-epoxide from the KH- anion. Pathway A was suggested by Dowd et al. based on studies on the model compound 2,4-dimethylnaphthol. Pathway B is not possible for 2,4-dimethylnaphthol but is a reasonable pathway for the oxidation of KH-.