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Proton NMR spectroscopy of the low-spin cyanide complex of horseradish peroxidase, HRPCN, in H20 solution was used to examine exchangeable resonances of functionally important amino acids in the heme pocket and their role in hydrogen-bonding networks, which have been proposed to facilitate peroxidase catalysis. Spectra were analyzed by use of nuclear Overhauser effect and saturation-transfer spectroscopies, along with consideration of paramagnetic shift and relaxation.Definitive assignments could be made in spite of the size of the proteins (42 kDa), its inherent paramagnetism (iron(III), 5 = */2), and the relatively few resolved resonances, suggesting that these NMR methods may be applicable to even larger heme proteins. The resonance identification was made for labile protons of the proximal His-170, distal His-42, and a heme peripheral contact, Tyr-185, which confirm the close similarity of the heme pocket stereochemistry of horseradish and cytochrome c peroxidases. The resonance assignments enabled determination of several key facets of the structure of the heme cavity of HRPCN. The proton taken up in concert with anionic ligand binding is shown to be present on the His-42 imidazole ring, forming a imidazolium side chain that hydrogen bonds sufficiently strongly to the nitrogen of the bound cyanide to induce a detectable isotope effect on the electronic structure of the heme. The strength of the hydrogen bond is confirmed by the stability of this network over the pH range 4-11. The labile ring proton of the proximal His-170 in HRPCN was located in a position that is consistent with an imidazolate description of the axial ligand but with the proton transferred to a nearby amino acid acceptor site less than 1 Á removed. The structural changes in the heme and proximal and distal amino acid residues upon ligand binding is discussed as a model for HRP compound II formation.

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Several fundamental requirements must be met so that NMR-based metabolomics and the related technique of metabonomics can be formally adopted into environmental monitoring and chemical risk assessment. Here we report an intercomparison exercise which has evaluated the effectiveness of 1H NMR metabolomics to generate comparable data sets from environmentally derived samples. It focuses on laboratory practice that follows sample collection and metabolite extraction, specifically the final stages of sample preparation, NMR data collection (500, 600, and 800 MHz), data processing, and multivariate analysis. Seven laboratories have participated from the U.S.A., Canada, U.K., and Australia, generating a total of ten data sets. Phase 1 comprised the analysis of synthetic metabolite mixtures, while Phase 2 investigated European flounder (Platichthys flesus) liver extracts from clean and contaminated sites. Overall, the comparability of data sets from the participating laboratories was good. Principal components analyses (PCA) of the individual data sets yielded ten highly similar scores plots for the synthetic mixtures, with a comparable result for the liver extracts. Furthermore, the same metabolic biomarkers that discriminated fish from clean and contaminated sites were discovered by all the laboratories. PCA of the combined data sets showed excellent clustering of the multiple analyses. These results demonstrate that NMR-based metabolomics can generate data that are sufficiently comparable between laboratories to support its continued evaluation for regulatory environmental studies.