MXAN analysis of the XANES energy region of a mononuclear copper complex: applications to bioinorganic systems

The near edge XAS spectra of the mononuclear copper complex [Cu(TMPA)(OH(2))](ClO(4))(2) (1) have been simulated using the multiple scattering edge simulation package MXAN (or Minuit XANes). These simulations, which employ the muffin-tin (MT) approximation, have been compared to simulations generated using the finite-difference method (FDM) to evaluate the effect of MT corrections. The sensitivity of the MXAN method was tested using structural models that included several different variations on the bond angles and bond distances for the first-shell atoms of 1. The sensitivity to small structural changes was also evaluated by comparing MXAN simulations of 1 and of structurally modified [Cu(TMPA)(L)](n)(+) complexes [where L = -O-(F(8)TPP)Fe(III), -F, -OPO(2)(O-p-nitrophenyl)Zn(II)(TMPA), and -NCMe] to the experimental data. The accuracy of the bond distances obtained from the MXAN simulations was then examined by comparison to the metrics of the crystal structures. The results show that MXAN can successfully extract geometric information from the edge structure of an XAS spectrum. The systematic application of MXAN to 1 indicates that this approach is sensitive to small structural changes in the molecule that are manifested in the XAS edge spectrum. These results represent the first step toward the application of this methodology to bioinorganic and biological systems.     

Quantitative determination of cetirizine enantiomers in guinea pig plasma, brain tissue and microdialysis samples using liquid chromatography/tandem mass spectrometry

Sensitive enantioselective liquid chromatographic assays using tandem mass spectrometric detection were developed and validated for the determination of S-cetirizine (S-CZE) and R-cetirizine (R-CZE) in guinea pig plasma, brain tissue, and microdialysis samples. Enantioselective separation was achieved on an alpha1-acid glycoprotein column within 14 min for all methods. A cetirizine analog, ucb 20028, was used as internal standard. Cetirizine and the internal standard were detected by multiple reaction monitoring using transitions m/z 389.1 --> 200.9 and 396.1 --> 276.1, respectively. The samples were prepared using protein precipitation with acetonitrile. For guinea pig plasma, the assay was linear over the range 0.25-5000 ng/mL for both S-CZE and R-CZE, with a lower limit of quantification (LLOQ) of 0.25 ng/mL. For the brain tissue and microdialysis samples, the assays were linear over the range 2.5-250 ng/g and 0.25-50 ng/mL, respectively, and the LLOQ values were 2.5 ng/g and 0.25 ng/mL, respectively. The intra- and inter-day precision values were < or =7.1% and < or =12.6%, respectively, and the intra- and inter-day accuracy varied by less than +/-8.0% and +/-6.0% of the nominal value, respectively, for both enantiomers in all the matrices investigated.     

On-line desalting and determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in microdialysis and plasma samples using column switching and liquid chromatography/tandem mass spectrometry

A sensitive and reproducible method for the determination of morphine and the metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) was developed. The method was validated for perfusion fluid used in microdialysis as well as for sheep and human plasma. A C18 guard column was used to desalt the samples before analytical separation on a ZIC HILIC (hydrophilic interaction chromatography) column and detection with tandem mass spectrometry (MS/MS). The mobile phases were 0.05% trifluoroacetic acid (TFA) for desalting and acetonitrile/5 mM ammonium acetate (70:30) for separation. Microdialysis samples (5 microL) were directly injected onto the system. The lower limits of quantification (LLOQ) for morphine, M3G and M6G were 0.50, 0.22 and 0.55 ng/mL, respectively, and the method was linear from LLOQ to 200 ng/mL. For plasma, a volume of 100 microL was precipitated with acetonitrile containing internal standards (deuterated morphine and metabolites). The supernatant was evaporated and reconstituted in 0.05% TFA before the desalting process. The LLOQs for sheep plasma were 2.0 and 3.1 ng/mL and the ranges were 2.0-2000 and 3.1-3100 ng/mL for morphine and M3G, respectively. For human plasma, the LLOQs were 0.78, 1.49 and 0.53 ng/mL and the ranges were 0.78-500, 1.49-1000 and 0.53-500 ng/mL for morphine, M3G and M6G, respectively.     

Density Functional Theory calculations on the magnetic properties of the model tyrosine radical-histidine complex mimicking tyrosyl radical Y<Subscript>D</Subscript><Superscript>·</Superscript> in photosystem II

Results of Density Functional Theory (DFT) theoretical investigations, which use a model tyrosyl (Tyr) radical and tyrosyl-histidine (Tyr-His) complex to mimick the Y _D ^· radical in Photosystem II (PSII) are presented and compared to experimental results from ¹⁵N Electron-Nuclear Double Resonance spectroscopy (ENDOR) studies of the τ nitrogen coupling from His-189 in the PSII Tyr-His complex. The DFT calculations are performed using an optimized geometry of the tyrosine radical and Tyr-His complex. The conformational space of the Tyr-His tandem is explored by varying the relative geometry of the two components; relevant parameters, such as the spin distribution on the phenoxy-ring carbons of the Tyr radical and the EPR hyperfine tensors, are calculated at each geometry and compared with the available experimental data. The isotropic ¹⁵N-ENDOR signal arising from spin delocalization on the His hydrogen-bonded to the PSII tyrosine radical is analyzed in terms of the DFT obtained parameters. The calculations of the g tensor using the Gauge Independent Atomic Orbital (GIAO) approach are presented and the influence of the geometry of the Tyr-His complex on the deviation of the g-tensor elements from the free electron values is discussed.     

Biosynthesis of the catalytic H-cluster of [FeFe] hydrogenase: the roles of the Fe–S maturase proteins HydE,HydF, and HydG

[FeFe] hydrogenases carry out the redox interconversion of protons and molecular hydrogen (2H⁺ + 2e⁻ ⇌ H₂) at a complex Fe–S active site known as the H-cluster. The H-cluster consists of a [4Fe–4S] subcluster, denoted here as [4Fe]_H, linked via a cysteine sulfur to an interesting organometallic [2Fe]_H subcluster thought to be the subsite where the catalysis occurs. This [2Fe]_H subcluster consists of two Fe atoms, linked with a bridging CO and a bridging SCH₂NHCH₂S azadithiolate (adt), with additional terminal CO and CN ligands bound to each Fe. Synthesizing such a complex organometallic unit is a fascinating problem in biochemistry, complicated by the toxic nature of both the CO and CN⁻ species and the relative fragility of the azadithiolate bridge. It has been known for a number of years that this complex biosynthesis is carried out by a set of three essential Fe–S proteins, HydE, HydF, and HydG. HydF is a GTPase, while HydE and HydG are both members of the large family of radical S-adenosylmethionine (rSAM) enzymes. In this perspective we describe the history of research and discovery concerning these three Fe–S “maturase” proteins and describe recent evidence for a sequential biosynthetic pathway beginning with the synthesis of a mononuclear organometallic [Fe(ii)(CO)₂CN(cysteine)] complex by the rSAM enzyme HydG and its subsequent activation by the second rSAM enzyme HydE to form a highly reactive Fe(i)(CO)₂(CN)S species. In our model a pair of these Fe(i)(CO)₂(CN)S units condense to form the [Fe(CO)₂(CN)S]₂ diamond core of the [2Fe]_H cluster, requiring only the installation of the central CH₂NHCH₂ portion of the azadithiolate bridge, whose atoms are all sourced from the amino acid serine. This final step likely occurs with an interplay of HydE and HydF, the details of which yet remain to be elucidated.

Fe–S cluster enzymes HydG, HydE, and HydF provide sequential assembly of the catalytic H-cluster of [FeFe] hydrogenase.     

Nature of the intermediate formed in the reduction of O(2) to H(2)O at the trinuclear copper cluster active site in native laccase

The multicopper oxidases contain at least four copper atoms and catalyze the four-electron reduction of O(2) to H(2)O at a trinuclear copper cluster. An intermediate, termed native intermediate, has been trapped by a rapid freeze-quench technique from Rhus vernicifera laccase when the fully reduced form reacts with dioxygen. This intermediate had been described as an oxygen-radical bound to the trinuclear copper cluster with one Cu site reduced. XAS, however, shows that all copper atoms are oxidized in this intermediate. A combination of EXAFS, multifrequency EPR, and VTVH MCD has been used to understand how this fully oxidized trinuclear Cu cluster relates to the fully oxidized resting form of the enzyme. It is determined that in the native intermediate all copper atoms of the cluster are bridged by the product of full O(2) reduction. In contrast, the resting form has one copper atom of the cluster (the T2 Cu) magnetically isolated from the others. The native intermediate decays to the resting oxidized form with a rate that is too slow to be in the catalytic cycle. Thus, the native intermediate appears to be the catalytically relevant fully oxidized form of the enzyme, and its role in catalysis is considered.     

Delocalization tunable by ligand substitution in [L2Al]n− complexes highlights a mechanism for strong electronic coupling

Ligand-based mixed valent (MV) complexes of Al(iii) incorporating electron donating (ED) and electron withdrawing (EW) substituents on bis(imino)pyridine ligands (I₂P) have been prepared. The MV states containing EW groups are both assigned as Class II/III, and those with ED functional groups are Class III and Class II/III in the (I₂P⁻)(I₂P²⁻)Al and [(I₂P²⁻)(I₂P³⁻)Al]²⁻ charge states, respectively. No abrupt changes in delocalization are observed with ED and EW groups and from this we infer that ligand and metal valence p-orbitals are well-matched in energy and the absence of LMCT and MLCT bands supports the delocalized electronic structures. The MV ligand charge states (I₂P⁻)(I₂P²⁻)Al and [(I₂P²⁻)(I₂P³⁻)Al]²⁻ show intervalence charge transfer (IVCT) transitions in the regions 6850–7740 and 7410–9780 cm⁻¹, respectively. Alkali metal cations in solution had no effect on the IVCT bands of [(I₂P²⁻)(I₂P³⁻)Al]²⁻ complexes containing –PhNMe₂ or –PhF₅ substituents. Minor localization of charge in [(I₂P²⁻)(I₂P³⁻)Al]²⁻ was observed when –PhOMe substituents are included.

Organo-aluminum mixed-valent complexes combine properties of both organic and transition element mixed-valent compounds. This supports delocalized electronic structures that are structurally and electronically tunable.     

Quantitative evaluation of sample application methods for semipreparative separations of basic proteins by two-dimensional gel electrophoresis

The use of cup-loading for sample application has become widely used in two-dimensional electrophoresis (2-DE) for resolution of basic proteins, but no side-by-side quantitative study has been published which compares cup-loading with the alternative passive and active rehydration methods to fully promote one type of loading method over another. Replicate 2-D gels from each loading method were quantitatively evaluated for gel-to-gel reproducibility using IPG 6-11 strips and semipreparative protein loads (300 microg). Gels were stained with SYPRO Ruby and analyzed with PDQuest. An inexpensive home-made assembly for cup-loading was used with the Protean IEF Cell for separation of whole cell extracts from the archaeon, Sulfolobus solfataricus. Cup-loading was determined to be far superior for IPG 6-11 separations than active or passive rehydration methods. Cup-loading consistently produced the greatest number of detectable spots, the best spot matching efficiency (56%), lowest spot quantity variations (28% coefficient of variation, CV), and the best-looking gels qualitatively. The least satisfactory results were obtained with active rehydration, followed closely by passive rehydration in off-line tubes. Passive rehydration experiments, performed using an on-line isoelectric focusing (IEF) tray, produced comparable spot numbers to cup-loading (84%), with 55% of the spots having higher intensity but 10% more spot quantity variance than cup-loading.     

Spectroscopic and density functional studies of the red copper site in nitrosocyanin: role of the protein in determining active site geometric and electronic structure

The electronic structure of the red copper site in nitrosocyanin is defined relative to that of the well understood blue copper site of plastocyanin by using low-temperature absorption, circular dichroism, magnetic circular dichroism, resonance Raman, EPR and X-ray absorption spectroscopies, combined with DFT calculations. These studies indicate that the principal electronic structure change in the red copper site is the sigma rather than the pi donor interaction of the cysteine sulfur with the Cu 3d(x2-y2) redox active molecular orbital (RAMO). Further, MCD data show that there is an increase in ligand field strength due to an increase in coordination number, whereas resonance Raman spectra indicate a weaker Cu-S bond. The latter is supported by the S K-edge data, which demonstrate a less covalent thiolate interaction with the RAMO of nitrosocyanin at 20% relative to plastocyanin at 38%. EXAFS results give a longer Cu-S(Cys) bond distance in nitrosocyanin (2.28 A) compared to plastocyanin (2.08 A) and also show a large change in structure with reduction of the red copper site. The red copper site is the only presently known blue copper-related site with an exogenous water coordinated to the copper. Density functional calculations reproduce the experimental properties and are used to determine the specific protein structure contributions to exogenous ligand binding in red copper. The relative orientation of the CuNNS and the CuSC(beta) planes (determined by the protein sequence) is found to be key in generating an exchangeable coordination position at the red copper active site. The exogenous water ligation at the red copper active site greatly increases the reorganization energy (by approximately 1.0 eV) relative to that of the blue copper protein site, making the red site unfavorable for fast outer-sphere electron transfer, while providing an exchangeable coordination position for inner-sphere electron transfer.